Therapeutic approaches for treating Parkinson&#39;s disease

ABSTRACT

The present invention relates to compositions and methods for the treatment of Parkinson&#39;s disease and related disorders. More specifically, the present invention relates to novel combinatorial therapies of Parkinson&#39;s disease and related disorders targeting the alpha-synuclein aggregation network. In particular, the invention relates to compounds which, alone or in combination(s), can effectively protect neuronal cells from alpha-synuclein aggregates. The invention also relates to methods of producing a drug or a drug combination for treating Parkinson&#39;s disease and to methods of treating Parkinson&#39;s disease or a related disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP2015/069783, filed Aug. 28, 2015, which isa continuation-in-part of U.S. Ser. No. 14/473,142, filed Aug. 29, 2014,now U.S. Pat. No. 9,248,111.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for thetreatment of Parkinson's disease and related disorders. Morespecifically, the present invention relates to novel combinatorialtherapies of Parkinson's disease and related disorders.

BACKGROUND OF THE INVENTION

Parkinsonism or Parkinsonian syndromes are a group of progressive,multicentric neurodegenerative disorders whose main features are tremorat rest, rigidity, bradykinesia and postural instability. Parkinson'sdisease (PD) is the most common form of Parkinsonism and the second mostcommon neurodegenerative disorder after Alzheimer's disease. Inindustrial countries, the prevalence of PD has been estimated atapproximately 0.3% of the general population, the elderly being the mostat risk (4% of the population over 80 are estimated to be affected). Themean age of onset is around 60 years, although early onset (as young as20 year old) can occur [1].

PD is often classified as a movement disorder. Rest tremor is the mostcommon and usually among the earliest symptoms to develop. Bradykinesiaalso usually appears in the early stages with difficulties performingtasks such as writing or getting dressed. Hyperkinetic movementdisorders have been reported as side-effects of some treatments ofParkinson's disease. In this regard, U.S. Pat. No. 5,952,389 patentdiscloses the use of acamprosate for alleviating levodopa-inducedhyperkinetic movement disorders. Limiting secondary effects of drugs is,however, distinct and remote from treating the disease or relatedsymptoms. Rigidity occurs and progresses to stiffness and resistance tomovement of the whole body, reducing the ability to move. In the latestages, the disease progresses to postural instability, leading toimpaired balance and frequent falls. Other motor symptoms such as gaitor swallowing disturbances can arise. If not treated, motor symptoms canlead to the patient being bedridden after an average of ten years [2,3].

In later stages of the disease, PD gives rise to many non-motor symptomswhich vary greatly individually. Disability is then greatly worsened bythe development of autonomic and neuropsychiatric disturbances.Disorders of speech, cognition, mood, behavior, and/or thought willdevelop, leading eventually to dementia. Other common symptoms includesensory, sleep and emotional problems. Those disorders decrease the lifeexpectancy of the individual affected and the mortality ratios arearound twice those of people without PD [2-4].

PD is an idiopathic disease and its pathophysiology also remains poorlyunderstood [4]. However, at least 5% of PD cases can be attributed togenetic variations. Mutations within genes such as SNCA(alpha-synuclein), PRKN (parkin), LRRK2 (leucine-rich repeat kinase 2),PINK1 (PTEN-induced putative kinase 1), DJ-1, ATP13A2 and eleven geneloci (PARK1-PARK11) have been associated with familial PD [5]. DJ-1 issuspected to be an ubiquitous redox-responsive cytoprotective proteinthereby confirming the pivotal role of oxidative stress in PD [6], thatis further evidenced by the protective role of Hypoxia Inducible Factor(HIF) in nigral dopaminergic cell protection against oxidative stress,mitochondrial dysfunction and iron homeostasis disturbance [7]. Apartfrom genetic factors, many environmental risk factors have been proposedto be involved in the onset of PD but none with undisputed evidence. Themost frequently replicated risk factor is exposure to metals, pesticidesor herbicides such as Agent Orange. On another hand, smoking andcaffeine consumption seems to protect individuals from PD [1].

The pathophysiology of PD is characterized by four features [4]:

(i) A synucleinopathy characterized by the abnormal accumulation ofalpha-synuclein protein into inclusions called Lewy bodies in the brain.The distribution of the Lewy bodies throughout the brain varies from oneindividual to another but is often directly associated with theexpression and degree of the clinical symptoms.(ii) Glutamate is the most abundant excitatory neurotransmitter in themammalian nervous system. Under pathological conditions, its abnormalaccumulation in the synaptic cleft leads to glutamate receptorsoveractivation that results in pathological processes and finally inneuronal cell death. This process, named excitotoxicity, is commonlyobserved in neuronal tissues during acute and chronic neurologicaldisorders. It is becoming evident that excitotoxicity is involved in thepathogenesis of Parkinson's disease.(iii) A dopaminergic activity deficiency due to the death ofdopamine-generating cells in the substantia nigra, a region of themidbrain. This results in a loss of muscle movement and tone control,leading to the motor symptoms of PD.(iv) Degeneration of NANC (non-adrenergic, non-cholinergic),serotonergic and cholinergic neurons also occurs in later stages of thedisease, leading to the non-motor symptoms of PD.

As no biological test is available, diagnosis of PD is mainly based onobservation of clinical symptoms and exclusion of other disorders withsimilar clinical features [3]. Postmortem confirmation is required for adefinitive diagnosis. Neurological examination by neuroimaging can beuseful to detect changes in dopaminergic neurons and to rule out otherdiseases. Positive therapeutic response to levodopa is another diagnosiscriterion. Once the diagnosis made, the progression and severity of thedisease is rated using a stages scale such as the Unified Parkinson'sDisease Rating Scale.

The most widely used treatment, especially at earlier stages, is thedopamine precursor, levodopa (L-DOPA) [8]. The drug brings the lackingneurotransmitter to the dopaminergic neurons, thus decreasing motorsymptoms. However, most of the drug is metabolized before to reach theblood brain barrier (BBB), causing a variety of side effects, especiallygastrointestinal effects (such as anorexia, nausea or vomiting),dyskinesia and psychiatric symptoms [9]. To prevent dyskinesiaphenomenon, L-DOPA is therefore usually given in combination withcarbidopa or benserazide (peripheral dopa decarboxylase inhibitors) andoften also with catechol-O-methyl transferase inhibitors such asentacapone. These drugs aim at preventing L-DOPA metabolism before toreach the brain, enhancing the activity of the drug [8]. Although lesseffective at improving motor symptoms, dopamine agonists such aspergolide, cabergoline, apomorphine or lisuride and monoamine oxidase-Binhibitors (involved in the catabolic breakdown of dopamine) such asselegiline or rasagiline are commonly used at early stages of thedisease. Although less effective, they may be useful at delaying the useof levodopa and thus the onset of dyskinesia [9].

Other drugs such as anticholinergics and nicotinic acetylcholinereceptor agonists may be useful but their efficacy for PD remains to beconfirmed [9]. Current research also focuses on neuroprotectivetreatments, but none of them provided evidence of improved degeneration.They target apoptosis (omigapil, CEP-1347), glutamate receptors,adenosine A2A receptor, calcium channels (isradipine), growth factors(GDNF), alpha-synuclein and inflammation [10].

Ongoing pharmaceutical research has shown a growing interest on genetherapy and neural transplantation [10].

WO 2009/133128, WO 2009/133141, WO 2009/133142, WO 2011/054759, WO2009/068668, WO 2009/153291 disclose potential treatments for severalneurodegenerative diseases, among which, PD.

PD remains so far an incurable disease and no effectivedisease-modifying treatment has been discovered yet. Therefore, currenttreatments aim at relieving symptoms and alleviate the slow progressionof the disease.

SUMMARY OF INVENTION

The present invention relates to new therapeutic methods andcompositions for treating Parkinsonism. The invention stems, inter alia,from the identification of drug combinations which provide improvedtherapeutic effect and clinical benefit to subjects having Parkinsonismcondition, particularly subjects having Parkinson's disease.

More particularly, an object of the invention relates to a compositionfor use in the treatment of Parkinsonism, particularly Parkinson'sdisease, comprising one, preferably at least two drugs selected fromacamprosate, baclofen, cinacalcet, mexiletine, sulfisoxazole, torasemideand tadalafil, or a salt, prodrug, derivative of any chemical purity, orsustained-release formulation thereof.

A further object of the invention is a method for treating Parkinsonism,particularly Parkinson's disease, in a subject in need thereof,comprising administering to the subject one, preferably at least twodrugs selected from acamprosate, baclofen, cinacalcet, mexiletine,sulfisoxazole, torasemide and tadalafil, or a salt, prodrug, derivativeof any chemical purity, or sustained-release formulation thereof.

Preferred examples of drug combinations for use in the inventioninclude, e.g., baclofen and acamprosate, baclofen and cinacalcet,mexiletine and cinacalcet, torasemide and baclofen, torasemide andsulfisoxazole, cinacalcet and tadalafil. In a particular embodiment, thecompositions and methods further comprise levodopa. In a anotherparticular embodiment, the compositions and methods further compriseselegiline or rasagiline. In more particular embodiment, when thecompositions and methods of the invention comprise a dopamine precursor,at least one compound selected from carbidopa, benserazide, tolcapone,entacapone, selegiline or rasagiline can be further used in saidcomposition or method.

The invention also relates to any pharmaceutical compositions per secomprising any drug combination selected from baclofen and acamprosate,baclofen and cinacalcet, mexiletine and cinacalcet, torasemide andbaclofen, torasemide and sulfisoxazole, cinacalcet and tadalafil.

The compositions in the invention may further comprise one or severalpharmaceutically acceptable carrier(s) or excipient(s), and they may beadministered repeatedly to the subject. Preferred compositions areadministered orally. Moreover, the drugs may be formulated oradministered together, separately or sequentially. The invention issuitable for treating Parkinsonism in any mammalian subject,particularly in a human subject, at any stage of the disease. Theinvention may be used e.g., to retard the development of the disease, toreduce, delay or prevent tremor, hypokinesia (e.g., bradykinesia,akinesia, rigidity), postural instability, and/or pain, and/or toincrease survival.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Effect of cinacalcet and mexiletine combination therapy againstglutamate toxicity on neuronal cortical cells. The glutamateintoxication is significantly prevented by the combination of cinacalcet(64 pM) and mexiletine (25.6 pM) whereas, at those concentrations,cinacalcet and mexiletine alone have no significant effect onintoxication. *: p<0.001, significantly different from glutamateintoxication; (ANOVA+Dunett's Post-Hoc test).

FIG. 2: Effect of sulfisoxazole and torasemide combination therapyagainst glutamate toxicity on neuronal cortical cells. The glutamateintoxication is significantly prevented by the combination ofsulfisoxazole (6.8 nM) and torasemide (400 nM) whereas, at thoseconcentrations, sulfisoxazole and torasemide alone have no significanteffect on intoxication.*: p<0.001, significantly different fromglutamate intoxication; (ANOVA+Dunett's Post-Hoc test).

FIG. 3: Effect of baclofen and acamprosate combination therapy againstglutamate toxicity on neuronal cortical cells. The glutamateintoxication is significantly prevented by the combination of baclofen(400 nM) and acamprosate (1.6 nM) whereas, at those concentrations,baclofen and acamprosate alone have no significant effect onintoxication. *: p<0.001, significantly different from glutamateintoxication; (ANOVA+Dunett's Post-Hoc test).

FIG. 4: Protective effect of baclofen and acamprosate combinationagainst ischemic injury. Whereas no significant protection is obtainedwhen baclofen (80 nM) or acamprosate (0.32 nM) are used alone, asignificant protection (*: p<0.0001) is observed for the combination ofthe two drugs, at the same concentrations.

FIG. 5: Protective effect of torasemide and sulfisoxazole combinationagainst ischemic injury. The combination of sulfisoxazole (1.36 nM) andtorasemide (80 nM) induces a significant protection (*: p<0.0001), 110%higher than the protection obtained using torasemide alone, whereas noprotection is obtained when sulfisoxazole is used alone.

FIG. 6: Protective effect of cinacalcet and mexiletine combinationagainst ischemic injury. No significant protection is observed whencinacalcet (64 pM) or mexiletine (25.6 pM) are used alone, whereas asignificant protection (*: p<0.0001) is observed for the combination ofthe two drugs, at the same concentrations.

FIG. 7: Effect of baclofen and acamprosate combination therapy against6-OHDA injury on dopaminergic neuronal cells. The protection increasescorrelatively with concentration of mixes. A significant protectiveeffect is observed with an increase in TH neurons survival by 34% withdose 1 (16 nM and 64 pM respectively), by 46% with dose 2 (80 nM and 144pM) and by 51% with dose 3 (400 nM and 1600 pM) (***: p<0.0001; *:p<0.001: significantly different from 6-OHDA intoxicated cells(ANOVA+Dunett's test)).

FIG. 8: Effect of baclofen and torasemide combination therapy against6-OHDA injury on dopaminergic neuronal cells. A significant protectiveeffect is observed with an increase in TH neurons survival by 50% withlow dose 1 (80 nM and 16 nM respectively), by 62% with the middle dose 2(240 nM and 48 nM) and by 58% with high dose 3 (720 nM and 144 nM) (***:p<0.0001: significantly different from 6-OHDA intoxicated cells(ANOVA+Dunett's test)).

FIG. 9: Effect of cinacalcet and mexiletine combination therapy against6-OHDA injury on dopaminergic neuronal cells. All the testedconcentrations afford a significant protection against 6-OHDA. Indeed, asignificant protective effect is observed with an increase in TH neuronssurvival by 36% with dose 1 (64 pM and 5 pM respectively), by 38% withthe dose 2 (64 pM and 26 pM) and by 46% with dose 3 (1600 pM and 64 pM)(***: p<0.0001; *: p<0.001: significantly different from 6-OHDAintoxicated cells (ANOVA+Dunett's test)).

FIG. 10: Initiation time test, effect of baclofen and acamprosatecombination therapy against 6-OHDA stereotaxic lesion in the leftsubstantia nigra pars compacta. Left paw: no significant change. Rightpaw: 6-OHDA injection strongly prolonged initiation time as a result ofdeath of neurons in the left substantia nigra. Baclofen-acamprosatetreatment strongly protects from 6-OHDA induced akinesia and this fromthe weakest dose 1 (baclofen-acamprosate dose 1: 0.6 mg/kg bid and 0.04mg/kg bid respectively; dose 2: 1.5 mg/kg bid and 0.1 mg/kg bid; dose 3:3.75 mg/kg bid and 0.25 mg/kg bid; ***: p<0.0001; **: p<0.001:significantly different from 6-OHDA intoxicated cells (ANOVA+Dunett'stest)); ns: no significant difference between data.

FIG. 11: Reaction time test, effect of baclofen and acamprosatecombination therapy against 6-OHDA stereotaxic lesion in the leftsubstantia nigra pars compacta. Left paw: no significant change. Rightpaw: 6-OHDA injection strongly prolonged reaction time as a result ofdeath of neurons in the left substantia nigra. Baclofen-acamprosatetreatment strongly protects from 6-OHDA induced akinesia and this fromthe weakest dose 1. Dose 2 and 3 almost fully alleviate 6-OHDA inducedakinesia (baclofen-acamprosate dose 1: 0.6 mg/kg bid and 0.04 mg/kg bid;dose 2: 1.5 mg/kg bid and 0.1 mg/kg bid; dose 3: 3.75 mg/kg bid and 0.25mg/kg bid; ***: p<0.0001: *: p<0.05: significantly different from 6-OHDAintoxicated cells (ANOVA+Dunett's test)); ns: no significant differencebetween data.

FIG. 12: Baclofen (BCL) and acamprosate (ACP) combination actssynergistically in protecting dopaminergic neuronal cells from 6-OHDAinjury. A: Baclofen, at a dose as low as 32 nM, when combined withacamprosate, at a dose as low as 10 pM, strongly protects dopaminergicneuron from 6-OHDA injury (+38% survival), while single drugs at thesame dose have low effect. B: Baclofen, 32 nM, combined with acamprosateat an even lower dose, 4 pM, also strongly protects cortical neuron from6-OHDA injury (+35%), while single drugs at the same dose have loweffect. ***: p<0.001; *: p<0.05, significantly different from 6-OHDAintoxicated cells (ANOVA+Dunett's test). S: synergy.

FIG. 13: Effect of acamprosate and cinacalcet combination therapyagainst 6-OHDA injury on dopaminergic neuronal cells. The testedconcentrations afford a significant protection against 6-OHDA. Indeed, asignificant protective effect is observed with an increase in TH neuronssurvival of 17% to 73% is observed. At each of these concentrations, thecombination exerts a synergistic protective effect. ***: p<0.001;significantly different from 6-OHDA intoxicated cells (ANOVA+Dunett'stest); 0: <0.05: significantly different from 6-OHDA intoxicated(Student's test).

FIG. 14: Effect of cinacalcet and tadalafil combination therapy against6-OHDA injury on dopaminergic neuronal cells. Combination therapyaffords a significant protection at different drug concentrations. Forthe different doses presented, drugs act synergistically to conferprotection. (***: p<0.001; **: p<0.01, significantly different from6-OHDA intoxicated cells (ANOVA+Dunett's test)).

FIG. 15: Cylinder test, effect of baclofen and acamprosate combinationtherapy against 6-OHDA stereotaxic lesion in the left substantia nigrapars compacta. 6-OHDA injection strongly decreased the number of doublecontacts as a result of death of neurons in the left substantia nigra(control, black bar). Baclofen-acamprosate treatment (3.75 mg/kg bid and0.25 mg/kg bid, respectively) strongly protects rats from 6-OHDA inducedakinesia, the number of contacts is significantly enhanced when comparedto control. ***: p<0.001; **: p<0.01: significantly different from6-OHDA intoxicated cells (ANOVA+Dunett's test)).

FIG. 16: Baclofen and acamprosate combination therapy (BCL-ACP; 3.75mg/kg bid and 0.25 mg/kg bid, respectively) protects dopaminergicneurons from death in vivo. A: BCL-ACP protects dopaminergic neuron cellbodies of subtantia nigra pars compacta (SN) from the 6-OHDA stereotaxiclesion in the left SN. The number of dopaminergic neurons is foundsignificantly increased in the left (intoxicated) part of SN of BCL-ACPtreated rats when compared to the number of neurons in the left(intoxicated) part of SN of non-treated animals. Such a neuroprotectiveeffect is not observed in L-DOPA treated rats. B: BCL-ACP protectsstriatal axonal projections of dopaminergic neurons after the 6-OHDAstereotaxic lesion in the left substantia nigra pars compacta (SN).BCL-ACP treatment significantly increased the density, in the leftstriatum, of dopaminergic neuronal terminals from the left part (6-OHDAintoxicated) of substantia nigra when compared to the left striatum ofnon-treated intoxicated rats (control, black bar). Such aneuroprotective effect is not observed for L-DOPA treated rats. OD:optical density related to the level of H³-mazindol labelling ofdopaminergic terminals in the striatum; labelling is expressed as the %of the OD obtained for the right part of striatum.***: p<0.001; **:p<0.01: significantly different from control; ns: not significantlydifferent from control (ANOVA+Dunett's test).

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide new therapeuticapproaches for treating Parkinsonism, more specifically Parkinson'sdisease. More particularly the invention discloses novel use of drugsand drug combinations and methods, which allow an effective correctionof such diseases and may be used in any mammalian subject.

Parkinsonism defines a group of progressive neurodegenerative disorderscharacterized by tremor at rest and/or bradykinesia associated withrigidity, postural instability, loss of postural reflexes, flexedposture, and/or the freezing phenomenon (when the feet are transiently“glued” to the ground). Examples of Parkinsonism conditions includeParkinson's disease, progressive supranuclear palsy, multiple systematrophy, cortical-basal ganglionic degeneration, diffuse Lewy bodydisease, Parkinson-dementia, X-linked dystonia-parkinsonism, andsecondary Parkinsonism (resulting from environmental etiology, e.g.,toxins, drugs, post encephalitic, brain tumors, head trauma, normalpressure hydrocephalus).

Parkinson's disease is the most common form of Parkinsonism. Parkinson'sdisease (“PD”) is a neurodegenerative disorder leading to motor andnon-motor manifestations and characterized by extensive degeneration ofdopaminergic neurons in the nigrostriatal system. The motormanifestations of PD are attributable to the degeneration ofdopaminergic neurons within the substantia nigra. They include tremor,hypokinesia (e.g., bradykinesia, akinesia, rigidity), posturalinstability, abnormal gait and swallowing disturbances. Non-motorsymptoms include autonomic and neuropsychiatric disturbances such asanosmia, or sleep abnormalities. Within the context of the invention,the term PD includes any of the above manifestations of the disease.

As used herein, “treatment” includes the therapy, prevention,prophylaxis, retardation or reduction of symptoms provoked by or of thecauses of Parkinsonism, preferably of Parkinson's disease. The termtreatment also designates a retardation or delayed onset of tremor, areduction of pain, a decrease or reduction of bradykinesia, akinesia,rigidity, postural instability, abnormal gait, anosmia, and/or sleepabnormalities, and/or an increase of survival. The term treatmentincludes in particular the control of disease progression and associatedmotor and non-motor symptoms. The term treatment particularly includesi) a protection against the toxicity caused by alpha-synuclein, or areduction or retardation of said toxicity, and/or ii) a protection ofdopaminergic neurons against the toxicity resulting from abnormalglutamate accumulation, oxidative stress, mitochondrial dysfunction orneuroinflammation, or a reduction or retardation of said toxicity, inthe treated subjects.

Within the context of this invention, the designation of a specific drugor compound is meant to include not only the specifically namedmolecule, but also any pharmaceutically acceptable salt, hydrate,derivative, isomer, racemate, conjugate, prodrug or derivative thereofof any chemical purity.

The term “combination or combinatorial treating/therapy” designates atreatment wherein at least two or more drugs are co-administered to asubject to cause a biological effect. In a combined therapy according tothis invention, the at least two drugs may be administered together orseparately, at the same time or sequentially. Also, the at least twodrugs may be administered through different routes and protocols. As aresult, although they may be formulated together, the drugs of acombination may also be formulated separately.

Several biological processes such as oxidative stress, mitochondrialdysfunction and neuroinflammation accompany accumulation of aggregatedalpha-synuclein which leads to the degeneration of dopaminergic neurons.On the other hand, abnormal accumulation of glutamate in synaptic cleftleads to the overactivation of glutamate receptors that results inpathological processes and finally in neuronal cell death. This processknown as excitotoxicity is now recognized as an important etiologicalfactor implicated in the development of Parkinson's disease.

The inventors were able to establish a network underlyingalpha-synuclein aggregation which is a major functional network affectedin Parkinson's disease. The inventors have identified functional modulescomposed of several target proteins, within the alpha-synucleinaggregation network. Such proteins are functionally relevant to thegenesis and control of Parkinson's disease and Parkinsonism, andrepresent valuable targets for therapies and particularly combinationtherapies.

Hence, the invention relates to the use of particular drugs which, aloneor preferentially in combination(s), modulate the above pathways totreat Parkinsonism, particularly Parkinson's disease.

In a particular embodiment, the present invention more specificallyrelates to compositions and methods using a drug combination thatinhibits the activity of at least two distinct proteins involved inalpha-synuclein aggregation network. The therapeutic approaches of theinvention are effective for the protection of neuronal cells,particularly for the protection of dopaminergic neurons in the midbrainand more particularly in the substantia nigra.

More particularly, the invention relates to a composition for use in thetreatment of Parkinsonism, particularly Parkinson's disease (PD),comprising at least two drugs selected from acamprosate, baclofen,cinacalcet, mexiletine, sulfisoxazole, torasemide and tadalafil, orsalts or prodrugs or derivatives of any purity or sustained releaseformulations thereof.

Indeed, the inventors have surprisingly found that these compounds showa protective activity against glutamate toxicity, which is a known causeof neuronal death in Parkinson's disease and Parkinsonism. The compoundsand combination therapies of the invention also show a protectiveactivity against ischemic stress which share common physiologicalfeatures with Parkinson's disease (notably mitochondrial dysfunction andoxidative stress). More particularly compounds of the present inventionare particularly efficient in vivo and in vitro against oxidative stresswhich is one component of alpha-synuclein toxicity for dopaminergicneurons.

The invention also relates to a method for the treatment ofParkinsonism, particularly Parkinson's disease (PD), comprisingadministering to a subject in need thereof at least two compoundsselected from acamprosate, baclofen, cinacalcet, mexiletine,sulfisoxazole, torasemide and tadalafil, or salts or prodrugs orderivatives of any purity or sustained release formulations thereof.

The term “prodrug” as used herein refers to any functional derivatives(or precursors) of a compound of the present invention, which, whenadministered to a biological system (e.g. a human organism), generatessaid compound as a result of e.g., spontaneous chemical reaction(s),enzyme catalyzed chemical reaction(s), and/or metabolic chemicalreaction(s). Prodrugs typically have the structure X-drug, wherein X isan inert carrier moiety and drug is the active compound. Prodrugs areusually inactive or less active than the resulting drug and can be used,for example, to improve the physicochemical properties of the drug, totarget the drug to a specific tissue, to improve the pharmacokinetic andpharmacodynamic properties of the drug and/or to reduce undesirable sideeffects. Some of the common functional groups that are amenable toprodrug design include, but are not limited to, carboxylic, hydroxyl,amine, phosphate/phosphonate and carbonyl groups. Prodrugs typicallyproduced via the modification of these groups include, but are notlimited to, esters, carbonates, carbamates, amides and phosphates.Specific technical guidance for the selection of suitable prodrugs isgeneral common knowledge [11-15]. Furthermore, the preparation ofprodrugs may be performed by conventional methods known by those skilledin the art. Methods which can be used to synthesize other prodrugs aredescribed in numerous reviews on the subject [11, 16-22]. For example,arbaclofen placarbil is listed in ChemID plus Advance database (website:chem.sis.nlm.nih.gov/chemidplus/) and arbaclofen placarbil is awell-known prodrug of baclofen [23, 24]. Specific examples of prodrugsof baclofen are given in Hanafi et al., 2011 [25], particularly baclofenesters and baclofen ester carbamates, which are of particular interestfor CNS targeting. Hence such prodrugs are particularly suitable forcompositions of this invention. Arbaclofen placarbil as mentioned beforeis also a well-known prodrug and may thus be used instead of baclofen incompositions of the invention. Other prodrugs of baclofen can be foundin the following patent applications: WO 2010/102071, US2009197958, WO2009/096985, WO 2009/061934, WO 2008/086492, US2009216037, WO2005/066122, US2011021571, WO 2003/077902, and WO 2010/120370.

Useful prodrugs for acamprosate such as pantoic acid ester neopentylsulfonyl esters, neopentyl sulfonyl esters prodrugs or maskedcarboxylate neopentyl sulfonyl ester prodrugs of acamprosate are notablylisted in WO 2009/033069, WO 2009/033061, WO 2009/033054 WO 2009/052191,WO 2009/033079, US20090099253, US20090069419, US20090082464,US20090082440, and US20090076147.

Prodrugs as described above can be used instead of the herein disclosedcompounds of the invention.

The term “derivative” of a compound includes any molecule that isfunctionally and structurally related to said compound, such as an acid,amide, ester, ether, acetylated variant, hydroxylated variant, or analkylated (C1-C6) variant of such a compound. The term “derivative” alsoincludes structurally related compound having lost one or moresubstituent as listed above. For example, homotaurine is a deacetylatedderivative of acamprosate. Preferred derivatives of a compound aremolecules having a substantial degree of similarity to said compound, asdetermined by known methods. Similar compounds along with their index ofsimilarity to a parent molecule can be found in numerous databases suchas PubChem (http://pubchem.ncbi.nlm.nih.gov/search/) or DrugBankWorldwide Websitc: drugbank.ca/). In a more preferred embodiment,derivatives should have a Tanimoto similarity index greater than 0.4,preferably greater than 0.5, more preferably greater than 0.6, even morepreferably greater than 0.7 with a parent drug. The Tanimoto similarityindex is widely used to measure the degree of structural similaritybetween two molecules. Tanimoto similarity index can be computed bysoftware such as the Small Molecule Subgraph Detector [26, 27] availableonline (see Worldwide Website: ebi.ac.uk/thornton-srv/software/SMSD/).Preferred derivatives should be both structurally and functionallyrelated to a parent compound, i.e., they should also retain at leastpart of the activity of the parent drug, more preferably they shouldhave a protective activity on dopaminergic neurons from 6-OHDA-inducedstress and/or glutamate toxicity and/or ischemic stress (as exemplifiedin the experimental part).

The term “derivative” also includes metabolites of a drug, e.g., amolecule which results from the (biochemical) modification(s) orprocessing of said drug after administration to an organism, usuallythrough specialized enzymatic systems, and which displays or retains abiological activity of the drug. Metabolites have been disclosed asbeing responsible for much of the therapeutic action of the parent drug.In a specific embodiment, a “metabolite” as used herein designates amodified or processed drug that retains at least part of the activity ofthe parent drug, preferably that has a protective activity ondopaminergic neurons from 6-OHDA-induced stress and/or glutamatetoxicity and/or ischemic stress. Examples of metabolites includehydroxylated forms of torasemide resulting from the hepatic metabolismof the drug [28].

The term “salt” refers to a pharmaceutically acceptable and relativelynon-toxic, inorganic or organic addition salt of a compound of thepresent invention. Pharmaceutical salt formation consists in pairing anacidic, basic or zwitterionic drug molecule with a counterion to createa salt version of the drug. A wide variety of chemical species can beused in neutralization reaction. Pharmaceutically acceptable salts ofthe invention thus include those obtained by reacting the main compound,functioning as a base, with an inorganic or organic acid to form a salt,for example, salts of acetic acid, nitric acid, tartric acid,hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonicacid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid orcitric acid. Pharmaceutically acceptable salts of the invention alsoinclude those in which the main compound functions as an acid and isreacted with an appropriate base to form, e.g., sodium, potassium,calcium, magnesium, ammonium, or choline salts. Though most of salts ofa given active principle are bioequivalents, some may have, amongothers, increased solubility or bioavailability properties. Saltselection is now a common standard operation in the process of drugdevelopment as taught by H. Stahl and C. G Wermuth in their handbook[29].

In a preferred embodiment, the designation of a compound is meant todesignate the compound per se, as well as any pharmaceuticallyacceptable salt, hydrate, isomer, racemate thereof.

Table 1 below provides non limiting examples of CAS number of compoundsfor use in the invention as well as of salt(s), derivatives,metabolites, and/or prodrugs of these compounds.

TABLE 1 Class or Tanimoto similarity Drug CAS Numbers index acamprosateand related compounds acamprosate 77337-76-9; 77337-73-6 NA homotaurine3687-18-1 0.73 Ehyl Dimethyl Ammonio / 0.77 Propane Sulfonate taurine107-35-7 0.5  baclofen and related compounds baclofen 1134-47-0;66514-99-6; NA 69308-37-8; 70206-22-3; 63701-56-4; 63701-55-3;28311-31-1 3-(p-chlorophenyl)-4- / Metabolite hydroxybutyric acidarbaclofen placarbil 847353-30-4 Prodrug mexiletine and relatedcompounds mexiletine 31828-71-4; 5370-01-4 6-hydroxymethylmexiletine53566-98-6 Metabolite 4-hydroxymexiletine 53566-99-7 Metabolite3-hydroxymexiletine (MHM) 129417-37-4 Metabolite N-hydroxymexiletine151636-18-9 Metabolite glucuronide sulfisoxazole and related compoundssulfisoxazole 127-69-5; 4299-60-9 N(4)-acetylsulfisoxazole 4206-74-0Metabolite sulfisoxazole acetyl 80-74-0 Prodrug sulfamethoxazole723-46-6 0.52 cinacalcet and related compounds cinacalcet 226256-56-0;364782-34-3 hydrocinnamic acid 501-52-0 Metabolite torasemide andrelated compounds torasemide 56211-40-6; 72810-59-4 hydroxytorasemide99300-68-2; 99300-67-1 Metabolites carboxytorasemide Metabolitetolbutamide 64-77-7 0.55 tadalafil and related compounds tadalafil171596-29-5; 171596-27-3; 171596-28-4; 629652-72-8 aminotadalafil385769-84-6  0.935

In a particular embodiment, a sustained-release formulation of thecompound is used.

The inventors have discovered that acamprosate, baclofen, cinacalcet,mexiletine, sulfisoxazole, torasemide and tadalafil are particularlyefficient in correcting alpha-synuclein aggregation molecular pathways.

As disclosed in the examples, molecules of the invention have a strong,unexpected effect on biological processes involved in Parkinsonism,particularly Parkinson's disease, and represent new therapeuticapproaches of the pathology. In particular, compositions of theinvention provide an unexpected protective effect against glutamatetoxicity. Furthermore, drugs and drug combinations of the inventionincrease the dopaminergic neuron survival under 6-OHDA-induced oxidativestress as well as ischemic stress, and induce a protective effect onmotor and non-motor manifestations of PD. These symptomatic improvementsare to be related to the actual neuronal protection of dopaminergicneurons which results, in vivo from the treatment with the compositionsof the invention. Thus, therapeutic approaches of the invention areeffective for the protection of neuronal cells, particularly for theprotection of dopaminergic neurons in the midbrain and more particularlyin the substantia nigra as shown in vivo.

In a particular embodiment, the present invention relates tocompositions and methods for treating Parkinsonism, particularly PDusing a compound selected from acamprosate, baclofen, cinacalcet,mexiletine, sulfisoxazole, torasemide and tadalafil.

Drug combinations that modulate the activity of at least two distinctproteins which are involved in the alpha-synuclein aggregation networkconstitute a particularly advantageous embodiment of the invention.Indeed, the inventors have observed that the above drugs, whenadministered in combination, act synergistically to efficiently protectdopaminergic neurons. In particular, compositions of the invention havean unexpected effect on glutamate toxicity, ischemia induced cell deathand oxidative stress. Such a strong and unexpected effect on biologicalprocesses involved in Parkinsonism, particularly PD, make these newcombinatorial therapeutic approaches of the pathology of particularinterest.

The invention thus also relates to compositions and methods for treatingParkinsonism, particularly PD, using at least two drugs selected fromacamprosate, baclofen, cinacalcet, mexiletine, sulfisoxazole, torasemideand tadalafil.

The compositions and methods of the invention lead to an improvement ofPD through their action on motor as well as non-motor symptoms of thedisease. Therapeutic approaches of the invention provide an efficientneuronal protection, particularly of dopaminergic neurons, againstoxidative stress, mitochondrial dysfunction, excitotoxicity damages,neuroinflammation or apoptosis. More particularly, they can provide aprotection of the substantia nigra neurons against the toxicity ofaggregated alpha-synuclein to reduce the rate or extent of dopaminergiccell loss and thereby affect the course of the disease progression.

In this regard, an object of this invention relates to a composition foruse in the treatment of Parkinsonism, particularly PD, comprising acompound selected from the group consisting of acamprosate, baclofen,cinacalcet, mexiletine, sulfisoxazole, torasemide, and tadalafil orsalts or prodrugs or derivatives of any purity or sustained releaseformulations thereof. In a preferred embodiment, the compositioncomprises torasemide or mexiletine, or a salt or prodrug or derivativeof any purity or sustained release formulation thereof.

The invention relates to a composition for use in the treatment ofParkinsonism, particularly PD, comprising at least two compoundsselected from acamprosate, baclofen, cinacalcet, mexiletine,sulfisoxazole, torasemide and tadalafil, or a salt, prodrug, derivativeof any chemical purity, or sustained release formulations thereof.

The invention relates to a composition for use in the treatment ofParkinsonism, particularly PD, comprising at least one compound selectedfrom acamprosate, cinacalcet, and torasemide, or a salt, prodrug,derivative of any chemical purity, or sustained release formulationsthereof, and at least one compound selected from baclofen, mexiletine,sulfisoxazole, and tadalafil or a salt, prodrug, derivative of anychemical purity, or sustained release formulations thereof.

More particularly, the invention relates to a composition comprising atleast one of the following drug combinations, for simultaneous,sequential or separate administration:

-   -   baclofen and acamprosate,    -   baclofen and cinacalcet,    -   cinacalcet and acamprosate,    -   mexiletine and cinacalcet,    -   torasemide and baclofen,    -   torasemide and sulfisoxazole, or    -   cinacalcet and tadalafil,        or salt(s) or prodrug(s) or derivative(s) of any purity or        sustained release formulations thereof for use in the treatment        of Parkinsonism, particularly PD.

The invention also relates to methods of treating Parkinsonism,particularly PD in a subject using any one of the above drugs orcompositions.

A particularly preferred composition or method of the invention usesbaclofen and acamprosate, or a salt, prodrug, derivative of any chemicalpurity, or sustained release formulations thereof.

Another preferred composition or method of the invention uses baclofenand torasemide, or a salt, prodrug, derivative of any chemical purity,or sustained release formulations thereof.

In another particular embodiment, the composition or method of theinvention uses acamprosate and cinacalcet, or salt(s) or prodrug(s) orderivative(s) of any purity or sustained release formulations, whereinthe daily dosage of acamprosate is equal or lower to 10 mg.

The invention also relates to baclofen, or a salt, prodrug, derivativeof any chemical purity, or sustained release formulations thereof, foruse in combination with acamprosate, or a salt, prodrug, derivative ofany chemical purity, or sustained release formulations thereof, for thetreatment of Parkinsonism, particularly PD, by combined, separate orsequential administration to a subject.

The invention also relates to the use of any of the above compositionfor use in protecting a subject in need thereof from the death ordegeneration of dopaminergic (DA) neurons. In a particular embodimentsaid subject is suffering from of Parkinsonism, and more particularlyfrom PD.

Preferred drug compositions of the invention therefore comprise 2, 3, 4or 5 distinct drugs, more preferably 2, 3 or 4 distinct drugs forcombinatorial treatment of Parkinsonism, particularly PD in a subject inneed thereof. In a preferred embodiment, the drugs of the invention areused in combination(s) for combined, separate or sequentialadministration, in order to provide the most effective effect.

The inventors further discovered that combination of at least one drugcombination selected from the group consisting of:

-   -   baclofen and acamprosate,    -   baclofen and cinacalcet,    -   mexiletine and cinacalcet,    -   cinacalcet and acamprosate,    -   torasemide and baclofen,    -   torasemide and sulfisoxazole, or    -   cinacalcet and tadalafil,        with a drug, different from previous, selected from drug        acamprosate, baclofen, cinacalcet, mexiletine, sulfisoxazole,        torasemide, and tadalafil and which enhances the therapeutic        effect of the binary combination and leads to even more        efficient compositions for use in the treatment of Parkinsonism,        particularly PD.

Thus, the invention also relates to a composition for use in thetreatment of Parkinsonism, particularly PD, comprising cinacalcet andacamprosate in a combination with a drug selected from drug baclofen,mexiletine, sulfisoxazole, torasemide and tadalafil or salts, prodrugs,derivatives or sustained release formulations thereof, for combined,separate or sequential administration.

The invention also relates to a composition for use in the treatment ofParkinsonism, particularly PD, comprising baclofen and acamprosate in acombination with a drug selected from cinacalcet, mexiletine,sulfisoxazole, torasemide and tadalafil or salts, prodrugs, derivativesor sustained release formulations thereof, for combined, separate orsequential administration.

The invention further relates to a composition for use in the treatmentof Parkinsonism, particularly PD, comprising baclofen and cinacalcet ina combination with a drug selected from acamprosate, mexiletine,sulfisoxazole, torasemide and tadalafil or salts, prodrugs, derivativesor sustained release formulations thereof, for combined, separate orsequential administration.

In an embodiment, the invention also relates to a composition for use inthe treatment of Parkinsonism, particularly PD, comprising mexiletineand cinacalcet in a combination with a drug selected from acamprosate,baclofen, sulfisoxazole, torasemide and tadalafil or salts, prodrugs,derivatives or sustained release formulations thereof, for combined,separate or sequential administration.

In another embodiment, the invention relates to a composition for use inthe treatment of Parkinsonism, particularly PD, comprising torasemideand baclofen in a combination with a drug selected from acamprosate,sulfisoxazole, cinacalcet, mexiletine and tadalafil or salts, prodrugs,derivatives or sustained release formulations thereof, for combined,separate or sequential administration.

In another embodiment, the invention further relates to a compositionfor use in the treatment of Parkinsonism, particularly PD, comprisingcinacalcet and tadalafil in a combination with a drug selected fromacamprosate, baclofen, mexiletine, sulfisoxazole and torasemide orsalts, prodrugs, derivatives or sustained release formulations thereof,for combined, separate or sequential administration.

The invention further relates to a composition for use in the treatmentof Parkinsonism, particularly PD, comprising torasemide andsulfisoxazole in a combination with a drug selected from acamprosate,baclofen, mexiletine, cinacalcet and tadalafil or salts, prodrugs,derivatives or sustained release formulations thereof, for combined,separate or sequential administration.

In a more particular embodiment, the invention relates to compositionsfor use in the treatment of Parkinsonism, particularly PD, comprising atleast one of the following drug combinations:

-   -   baclofen and cinacalcet and mexiletine,    -   cinacalcet and acamprosate and mexiletine,    -   baclofen and acamprosate and cinacalcet,    -   baclofen and acamprosate and torasemide,    -   baclofen and acamprosate and mexiletine,    -   baclofen and acamprosate and tadalafil,    -   torasemide and baclofen and cinacalcet,    -   cinacalcet and tadalafil and mexiletine,    -   cinacalcet and tadalafil and acamprosate, or    -   cinacalcet and tadalafil and baclofen,        or salts, prodrugs, derivatives or sustained release        formulations thereof, for combined, separate or sequential        administration.

A further object of this invention resides in the use of a compositionas defined above for the manufacture of a medicament for treatingParkinsonism, particularly PD.

Another object of the invention relates to the use a composition asdefined above for the manufacture of a medicament for protecting asubject in need thereof from the death or degeneration of DA neurons.

As indicated previously, in a combination therapy of this invention, thecompounds or drugs may be formulated together or separately, andadministered together, separately or sequentially.

A further object of the invention is a method of treating Parkinsonism,particularly PD, the method comprising simultaneously, separately orsequentially administering to a subject in need thereof an effectiveamount of a composition as disclosed above.

In this regard, a particular object of the invention is a method oftreating Parkinsonism, particularly PD, the method comprisingsimultaneously, separately or sequentially administering to a subject inneed thereof an effective amount of a drug combination as defined above.

In a preferred embodiment, the invention relates to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of a composition of at least one compound selectedfrom the group consisting of acamprosate, baclofen, cinacalcet,mexiletine, sulfisoxazole, torasemide and tadalafil.

In a more preferred embodiment, the invention relates to a method oftreating Parkinsonism, particularly PD in a subject in need thereof,comprising administering simultaneously, separately or sequentially tothe subject an effective amount of a combination of at least twocompounds selected from the group consisting of acamprosate, baclofen,cinacalcet, mexiletine, sulfisoxazole, torasemide and tadalafil.

In an even more preferred embodiment, the invention relates to a methodof treating Parkinsonism, particularly PD in a subject in need thereof,comprising administering simultaneously, separately or sequentially tothe subject an effective amount of a combination of at least one of thefollowing drug combinations, or salts, prodrugs derivatives or sustainedrelease formulation thereof:

-   -   baclofen and acamprosate,    -   baclofen and cinacalcet,    -   cinacalcet and acamprosate,    -   mexiletine and cinacalcet,    -   torasemide and baclofen,    -   torasemide and sulfisoxazole, or    -   cinacalcet and tadalafil.

In a another preferred embodiment, the invention relates to a method oftreating Parkinsonism, particularly PD in a subject in need thereof,comprising administering simultaneously, separately or sequentially tothe subject an effective amount of a combination of at least one of thefollowing drug combinations, or salts, prodrugs derivatives or sustainedrelease formulation thereof:

-   -   baclofen and cinacalcet and mexiletine,    -   cinacalcet and acamprosate and mexiletine,    -   baclofen and acamprosate and cinacalcet,    -   baclofen and acamprosate and torasemide,    -   baclofen and acamprosate and mexiletine,    -   baclofen and acamprosate and tadalafil,    -   torasemide and baclofen and cinacalcet,    -   cinacalcet and tadalafil and mexiletine,    -   cinacalcet and tadalafil and acamprosate, or    -   cinacalcet and tadalafil and baclofen.

In another embodiment, the invention relates to a method of protecting asubject in need thereof from the death or degeneration of DA neurons,said method comprising administering any of the above compositions.

The compositions of the invention typically comprise one or severalpharmaceutically acceptable carriers or excipients. Also, for use in thepresent invention, the drugs or compounds are usually mixed withpharmaceutically acceptable excipients or carriers.

In this regard, a further object of this invention is a method ofpreparing a pharmaceutical composition, the method comprising mixing theabove compounds or compound combinations in an appropriate excipient orcarrier.

Although very effective in vitro and in vivo, depending on the subjector specific condition, the above methods, compositions or combinationtherapies may further be used in conjunction or association orcombination with additional drugs or treatments.

Additional therapies used in conjunction with drug(s) or drug(s)combination(s) according to the present invention, may comprise one ormore drug(s) that ameliorate symptoms of PD, one or more drug(s) thatcould be used for palliative treatment of PD or one or more drug(s)currently evaluated in the frame of clinical trials for treatingParkinson's disease.

Therefore, compositions of the invention can be combined withdopaminergic drugs such as dopamine precursors (preferably levodopa,melevodopa), dopamine receptor agonists (preferably talipexole,piribedil, rotigotine, bromocriptine, pergolide, cabergoline, lisuride,pramipexole, ropinirole or apomorphine) or inhibitors ofdopamine-metabolizing enzymes (preferably selegiline, rasagiline).

Compositions of the invention can also be combined with other knowntreatments for PD, adjunctive treatments for PD, or treatment of thenon-motor symptoms of PD or, preferablymonosialotetrahexosylganglioside, citicoline, droxidopa mazaticol,promethazine, quetiapine, procyclidine, orphenadrine, domperidone,benzatropine, trihexyphenidyl, biperiden, clozapine, desipramine,citalopram, nortriptyline, paroxetine, atomoxetine, venlafaxine,amantadine, donepezil, rivastigmine or memantine.

Such use of methods, compositions or combinations of the invention withthe above mentioned therapies would permit the lowering of therapeuticdoses of the concerned drugs and thus would reduce, delay or avoid knownside effects associated with these drugs, for instance peak-dosedyskinesia which is observed in patients treated with levodopa.

In this regard, a further object of this invention relates to acomposition for use in the treatment of Parkinsonism, particularly PD,comprising a composition as defined above, in combination with at leastone compound selected from the group consisting of levodopa, melevodopa,talipexole, piribedil, rotigotine, bromocriptine, pergolide,cabergoline, lisuride, pramipexole, ropinirole, apomorphine, selegiline,rasagiline, monosialotetrahexosylganglioside, citicoline, droxidopamazaticol, promethazine, quetiapine, procyclidine, orphenadrine,domperidone, benzatropine, trihexyphenidyl, biperiden, clozapine,desipramine, citalopram, nortriptyline, paroxetine, atomoxetine,venlafaxine, amantadine, donepezil, rivastigmine and memantine, or saltsor prodrugs or derivatives of any purity or sustained releaseformulations thereof.

Hence, a particular embodiment of this invention relates to acomposition for use in the treatment of Parkinsonism, particularly PD,comprising at least one compound selected from the group consisting ofacamprosate, baclofen, cinacalcet, mexiletine, sulfisoxazole,torasemide, or tadalafil or salt(s), prodrug(s), derivative(s) of anychemical purity, or sustained-release formulation(s) thereof, incombination with at least one compound selected from the groupconsisting of levodopa, melevodopa, talipexole, piribedil, rotigotine,bromocriptine, pergolide, cabergoline, lisuride, pramipexole,ropinirole, apomorphine, selegiline, rasagiline,monosialotetrahexosylganglioside, citicoline, droxidopa mazaticol,promethazine, quetiapine, procyclidine, orphenadrine, domperidone,benzatropine, trihexyphenidyl, biperiden, clozapine, desipramine,citalopram, nortriptyline, paroxetine, atomoxetine, venlafaxine,amantadine, donepezil, rivastigmine and memantine, or salts or prodrugsor derivatives of any purity or sustained release formulations thereof.

A preferred embodiment of the invention relates to a composition for usein the treatment of Parkinsonism, particularly PD comprising at leastone of the following drug combinations:

-   -   baclofen and levodopa,    -   torasemide and levodopa,    -   sulfisoxazole and levodopa,    -   mexiletine and levodopa,    -   cinacalcet and levodopa,    -   tadalafil and levodopa,    -   baclofen and selegiline or rasagiline,    -   torasemide and selegiline or rasagiline,    -   sulfisoxazole and selegiline or rasagiline,    -   mexiletine and selegiline or rasagiline,    -   cinacalcet and selegiline or rasagiline, or    -   tadalafil and selegiline or rasagiline,        or salts or prodrugs or derivatives of any purity or sustained        release formulations thereof, for combined, separate or        sequential administration.

The invention also relates to a composition per se comprising at leastone of the following drug combinations:

-   -   baclofen and levodopa,    -   torasemide and levodopa,    -   sulfisoxazole and levodopa,    -   mexiletine and levodopa,    -   cinacalcet and levodopa,    -   tadalafil and levodopa,    -   baclofen and selegiline or rasagiline,    -   torasemide and selegiline or rasagiline,    -   sulfisoxazole and selegiline or rasagiline,    -   mexiletine and selegiline or rasagiline,    -   cinacalcet and selegiline or rasagiline, or    -   tadalafil and selegiline or rasagiline        or salts or prodrugs or derivatives of any purity or sustained        release formulations thereof, for combined, separate or        sequential administration.

In an embodiment, the invention relates also to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of a combination of at least two compounds selectedfrom the group consisting of acamprosate, baclofen, cinacalcet,mexiletine, sulfisoxazole, torasemide or tadalafil, in combination withat least one compound selected from the group consisting of levodopa,melevodopa, talipexole, piribedil, rotigotine, bromocriptine, pergolide,cabergoline, lisuride, pramipexole, ropinirole, apomorphine, selegiline,rasagiline, monosialotetrahexosylganglioside, citicoline, droxidopamazaticol, promethazine, quetiapine, procyclidine, orphenadrine,domperidone, benzatropine, trihexyphenidyl, biperiden, clozapine,desipramine, citalopram, nortriptyline, paroxetine, atomoxetine,venlafaxine, amantadine, donepezil, rivastigmine and memantine, or saltsor prodrugs or derivatives of any purity or sustained releaseformulations thereof.

In another embodiment, the invention relates to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of baclofen and acamprosate or salts, prodrugs,derivatives or sustained release formulations thereof, in combinationwith at least one compound selected from the group consisting oflevodopa, melevodopa, talipexole, piribedil, rotigotine, bromocriptine,pergolide, cabergoline, lisuride, pramipexole, ropinirole, apomorphine,selegiline, rasagiline, monosialotetrahexosylganglioside, citicoline,droxidopa mazaticol, promethazine, quetiapine, procyclidine,orphenadrine, domperidone, benzatropine, trihexyphenidyl, biperiden,clozapine, desipramine, citalopram, nortriptyline, paroxetine,atomoxetine, venlafaxine, amantadine, donepezil, rivastigmine andmemantine, or salts or prodrugs or derivatives of any purity orsustained release formulations thereof.

In another embodiment, the invention relates to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of mexiletine and cinacalcet or salts, prodrugs,derivatives or sustained release formulations thereof, in combinationwith at least one compound selected from the group consisting oflevodopa, melevodopa, talipexole, piribedil, rotigotine, bromocriptine,pergolide, cabergoline, lisuride, pramipexole, ropinirole, apomorphine,selegiline, rasagiline, monosialotetrahexosylganglioside, citicoline,droxidopa mazaticol, promethazine, quetiapine, procyclidine,orphenadrine, domperidone, benzatropine, trihexyphenidyl, biperiden,clozapine, desipramine, citalopram, nortriptyline, paroxetine,atomoxetine, venlafaxine, amantadine, donepezil, rivastigmine andmemantine, or salts or prodrugs or derivatives of any purity orsustained release formulations thereof.

In another embodiment, the invention relates to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of torasemide and baclofen or salts, prodrugs,derivatives or sustained release formulations thereof, in combinationwith at least one compound selected from the group consisting oflevodopa, melevodopa, talipexole, piribedil, rotigotine, bromocriptine,pergolide, cabergoline, lisuride, pramipexole, ropinirole, apomorphine,selegiline, rasagiline, monosialotetrahexosylganglioside, citicoline,droxidopa mazaticol, promethazine, quetiapine, procyclidine,orphenadrine, domperidone, benzatropine, trihexyphenidyl, biperiden,clozapine, desipramine, citalopram, nortriptyline, paroxetine,atomoxetine, venlafaxine, amantadine, donepezil, rivastigmine andmemantine, or salts or prodrugs or derivatives of any purity orsustained release formulations thereof.

In another embodiment, the invention relates to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of sulfisoxazole and torasemide or salts, prodrugs,derivatives or sustained release formulations thereof, in combinationwith at least one compound selected from the group consisting oflevodopa, melevodopa, talipexole, piribedil, rotigotine, bromocriptine,pergolide, cabergoline, lisuride, pramipexole, ropinirole, apomorphine,selegiline, rasagiline, monosialotetrahexosylganglioside, citicoline,droxidopa mazaticol, promethazine, quetiapine, procyclidine,orphenadrine, domperidone, benzatropine, trihexyphenidyl, biperiden,clozapine, desipramine, citalopram, nortriptyline, paroxetine,atomoxetine, venlafaxine, amantadine, donepezil, rivastigmine andmemantine, or salts or prodrugs or derivatives of any purity orsustained release formulations thereof.

In another embodiment, the invention relates to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of cinacalcet and acamprosate or salts, prodrugs,derivatives or sustained release formulations thereof, in combinationwith at least one compound selected from the group consisting oflevodopa, melevodopa, talipexole, piribedil, rotigotine, bromocriptine,pergolide, cabergoline, lisuride, pramipexole, ropinirole, apomorphine,selegiline, rasagiline, monosialotetrahexosylganglioside, citicoline,droxidopa mazaticol, promethazine, quetiapine, procyclidine,orphenadrine, domperidone, benzatropine, trihexyphenidyl, biperiden,clozapine, desipramine, citalopram, nortriptyline, paroxetine,atomoxetine, venlafaxine, amantadine, donepezil, rivastigmine andmemantine, or salts or prodrugs or derivatives of any purity orsustained release formulations thereof.

In another embodiment, the invention relates to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of baclofen and cinacalcet or salts, prodrugs,derivatives or sustained release formulations thereof, in combinationwith at least one compound selected from the group consisting oflevodopa, melevodopa, talipexole, piribedil, rotigotine, bromocriptine,pergolide, cabergoline, lisuride, pramipexole, ropinirole, apomorphine,selegiline, rasagiline, monosialotetrahexosylganglioside, citicoline,droxidopa mazaticol, promethazine, quetiapine, procyclidine,orphenadrine, domperidone, benzatropine, trihexyphenidyl, biperiden,clozapine, desipramine, citalopram, nortriptyline, paroxetine,atomoxetine, venlafaxine, amantadine, donepezil, rivastigmine andmemantine, or salts or prodrugs or derivatives of any purity orsustained release formulations thereof.

In another embodiment, the invention relates to a method of treatingParkinsonism, particularly PD in a subject in need thereof, comprisingadministering simultaneously, separately or sequentially to the subjectan effective amount of cinacalcet and tadalafil or salts, prodrugs,derivatives or sustained release formulations thereof, in combinationwith at least one compound selected from the group consisting oflevodopa, melevodopa, talipexole, piribedil, rotigotine, bromocriptine,pergolide, cabergoline, lisuride, pramipexole, ropinirole, apomorphine,selegiline, rasagiline, monosialotetrahexosylganglioside, citicoline,droxidopa mazaticol, promethazine, quetiapine, procyclidine,orphenadrine, domperidone, benzatropine, trihexyphenidyl, biperiden,clozapine, desipramine, citalopram, nortriptyline, paroxetine,atomoxetine, venlafaxine, amantadine, donepezil, rivastigmine andmemantine, or salts or prodrugs or derivatives of any purity orsustained release formulations thereof.

In a preferred embodiment, the compositions of this invention, for usein the treatment of Parkinsonism, particularly PD, comprise at least oneof the following drug combinations, or salts, prodrugs, derivatives orsustained release formulations thereof, the drugs in each of saidcombinations being for combined, separate or sequential administration:

-   -   baclofen and acamprosate and levodopa,    -   mexiletine and cinacalcet and levodopa,    -   torasemide and baclofen and levodopa,    -   baclofen and cinacalcet and levodopa,    -   cinacalcet and acamprosate and levodopa,    -   sulfisoxazole and torasemide and levodopa,    -   cinacalcet and tadalafil and levodopa,    -   baclofen and acamprosate and selegiline or rasagiline,    -   mexiletine and cinacalcet and selegiline or rasagiline,    -   torasemide and baclofen and selegiline or rasagiline,    -   baclofen and cinacalcet and selegiline or rasagiline,    -   cinacalcet and acamprosate and selegiline or rasagiline,    -   sulfisoxazole and torasemide and selegiline or rasagiline, or    -   cinacalcet and tadalafil and selegiline or rasagiline.

The invention also relates to a composition per se comprising at leastone of the following drug combinations, or salts, prodrugs, derivativesor sustained release formulations thereof, the drugs in each of saidcombinations being for simultaneous, separate or sequentialadministration:

-   -   baclofen and acamprosate and levodopa,    -   mexiletine and cinacalcet and levodopa,    -   torasemide and baclofen and levodopa,    -   baclofen and cinacalcet and levodopa,    -   cinacalcet and acamprosate and levodopa,    -   sulfisoxazole and torasemide and levodopa,    -   cinacalcet and tadalafil and levodopa,    -   baclofen and acamprosate and selegiline or rasagiline,    -   mexiletine and cinacalcet and selegiline or rasagiline,    -   torasemide and baclofen and selegiline or rasagiline,    -   baclofen and cinacalcet and selegiline or rasagiline,    -   cinacalcet and acamprosate and selegiline or rasagiline,    -   sulfisoxazole and torasemide and selegiline or rasagiline, or    -   cinacalcet and tadalafil and selegiline or rasagiline.

In another preferred embodiment, the invention relates to compositionsof this invention, for use in the treatment of Parkinsonism,particularly PD, comprise at least one of the following drugcombinations, or salts, prodrugs, derivatives or sustained releaseformulations thereof, the drugs in each of said combinations being forcombined, separate or sequential administration:

-   -   baclofen, cinacalcet, mexiletine and levodopa,    -   cinacalcet, acamprosate, mexiletine and levodopa,    -   baclofen, acamprosate, cinacalcet and levodopa,    -   baclofen, acamprosate, torasemide and levodopa,    -   baclofen, acamprosate, mexiletine, and levodopa,    -   baclofen, acamprosate, tadalafil, and levodopa,    -   torasemide, baclofen, cinacalcet and levodopa,    -   cinacalcet, tadalafil, mexiletine, and levodopa,    -   cinacalcet, tadalafil, acamprosate, and levodopa,    -   cinacalcet, tadalafil, baclofen, and levodopa    -   baclofen, cinacalcet, mexiletine and selegiline or rasagiline,    -   cinacalcet, acamprosate, mexiletine and selegiline or        rasagiline,    -   baclofen, acamprosate, cinacalcet and selegiline or rasagiline,    -   baclofen, acamprosate, torasemide and selegiline or rasagiline,    -   baclofen, acamprosate, mexiletine, and selegiline or rasagiline,    -   baclofen, acamprosate, tadalafil, and selegiline or rasagiline,    -   torasemide, baclofen, cinacalcet and selegiline or rasagiline,    -   cinacalcet, tadalafil, mexiletine, and selegiline or rasagiline,    -   cinacalcet, tadalafil, acamprosate, and selegiline or        rasagiline, or    -   cinacalcet, tadalafil, baclofen, and selegiline or rasagiline.

In a particular embodiment, when compositions or combination therapiesof the invention comprise dopamine precursor, they can be furthercombined with at least one compound selected from peripheral dopadecarboxylase inhibitors, catechol-O-methyl transferase inhibitors ormonoamine oxidase inhibitors. More particularly, when compositions orcombination therapies of the invention comprise a dopamine precursor,they can be further combined with at least one compound selected fromcarbidopa, benserazide, tolcapone, entacapone, selegiline or rasagiline.

A further object of this invention resides in the use of a compositionas defined above for the manufacture of a medicament for treatingParkinsonism, particularly PD.

In another embodiment, compositions or combination therapies of theinvention can be used in conjunction with surgical therapy for PD suchas deep brain stimulation. More particularly, surgical therapies aredeep brain stimulation of the subthalamic nucleus or of the globuspallidus interna.

In this regard, the invention relates to a composition comprising atleast one compound selected from the group consisting of acamprosate,baclofen, cinacalcet, mexiletine, sulfisoxazole, torasemide andtadalafil or salt(s), prodrug(s), derivative(s) of any chemical purity,or sustained-release formulation(s) thereof, for use in combination withdeep brain stimulation of the subthalamic nucleus or of the globuspallidus interna, in the treatment of Parkinsonism, particularly PD.

PD motor symptoms can develop lately when the dopaminergic denervationof the striatum and lose of substantia nigra dopaminergic neurons arealready widely occurring. Thus, the treatment of PD before motorsymptoms appearance and in prevention is essential in order to alter theprogression and course of the disease.

In this regard, in a preferred embodiment, any of the above methods,compositions or combination therapies can be used for the prevention,prophylaxis or retardation of symptoms provoked by or of the causes ofPD.

The combination of early detection of non-motor symptoms, mostparticularly anosmia, with imaging techniques (Single-photon emissioncomputed technology, Positron Emission Tomography) to assess changes instriatal dopamine transporter may be a suitable approach to identify atrisk PD patients prior to the appearance of motor symptoms, thusallowing early start of neuroprotective therapy.

Some PD cases can be attributed to mutations within genes such as SNCA(alpha-synuclein), PRKN (parkin), LRRK2 (leucine-rich repeat kinase 2),PINK1 (PTEN-induced putative kinase 1), DJ-1 and ATP13A2 and eleven geneloci (PARK1-PARK11). In this regard, in a particular embodiment, theinvention relates to the use of the above methods, compositions orcombination therapies for the treatment of PD in a subject having amutation in at least one of the following genes: SNCA, PRKN, LRRK2,PINK1, DJ-1, ATP13A2 and PARK1 to PARK11.

High concentrations exposure or chronic exposure to metals such asmanganese, copper or lead, or chemicals, such as pesticides (e.g.paraquat, rotenone and maneb), are likely to cause Parkinsonism,particularly PD. In this regard, in a particular embodiment, theinvention relates to the use of the above methods, compositions orcombination therapies in the treatment of Parkinsonism, particularly PD,in a subject exposed, suspected to have been exposed or at risk of beingexposed to chemicals or metals known to be risk factors for developingPD or related disorders.

In a preferred embodiment, the above methods, compositions orcombination therapies can be used in a subject who is at risk ofdeveloping PD or symptoms associated with PD.

In another preferred embodiment, the above methods, compositions orcombination therapies can be used for protecting a subject who is atrisk of developing PD or symptoms associated with PD, from the death ordegeneration of DA neurons.

Therapy according to the invention may be provided at home, the doctor'soffice, a clinic, a hospital's outpatient department, or a hospital, sothat the doctor can observe the therapy's effects closely and make anyadjustments that are needed.

The duration of the therapy depends on the stage of the disease beingtreated, age and condition of the patient, and how the patient respondsto the treatment. The dosage, frequency and mode of administration ofeach component of the combination can be controlled independently. Forexample, one drug may be administered orally while the second drug maybe administered intramuscularly. Combination therapy may be given inon-and-off cycles that include rest periods so that the patient's bodyhas a chance to recovery from any as yet unforeseen side-effects. Thedrugs may also be formulated together such that one administrationdelivers all drugs.

The administration of each drug of the combination may be by anysuitable means that results in a concentration of the drug that,combined with the other component, is able to ameliorate the patientcondition or efficiently treat the disease or disorder.

While it is possible for the drugs of the combination to be administeredas the pure chemical, it is preferable to present them as apharmaceutical composition, also referred to in this context aspharmaceutical formulation. Possible compositions include those suitablefor oral, rectal, topical (including transdermal, buccal andsublingual), or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration.

More commonly these pharmaceutical formulations are prescribed to thepatient in “patient packs” containing a number dosing units or othermeans for administration of metered unit doses for use during a distincttreatment period in a single package, usually a blister pack. Patientpacks have an advantage over traditional prescriptions, where apharmacist divides a patient's supply of a pharmaceutical from a bulksupply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in traditionalprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions. Thus, theinvention further includes a pharmaceutical formulation, as hereinbefore described, in combination with packaging material suitable forsaid formulations. In such a patient pack the intended use of aformulation for the combination treatment can be inferred byinstructions, facilities, provisions, adaptations and/or other means tohelp using the formulation most suitably for the treatment. Suchmeasures make a patient pack specifically suitable for and adapted foruse for treatment with the combination of the present invention.

The drug may be contained, in any appropriate amount, in any suitablecarrier substance. The drug may be present in an amount of up to 99% byweight of the total weight of the composition. The composition may beprovided in a dosage form that is suitable for the oral, parenteral(e.g., intravenously, intramuscularly), rectal, cutaneous, nasal,vaginal, inhalant, skin (patch), or ocular administration route. Thus,the composition may be in the form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, osmoticdelivery devices, suppositories, enemas, injectables, implants, sprays,or aerosols.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20th ed.), [30] and Encyclopedia ofPharmaceutical Technology [31]).

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

The controlled release formulations include (i) formulations that createa substantially constant concentration of the drug within the body overan extended period of time; (ii) formulations that after a predeterminedlag time create a substantially constant concentration of the drugwithin the body over an extended period of time; (iii) formulations thatsustain drug action during a predetermined time period by maintaining arelatively, constant, effective drug level in the body with concomitantminimization of undesirable side effects associated with fluctuations inthe plasma level of the active drug substance; (iv) formulations thatlocalize drug action by, e.g., spatial placement of a controlled releasecomposition adjacent to or in the diseased tissue or organ; and (v)formulations that target drug action by using carriers or chemicalderivatives to deliver the drug to a particular target cell type.

Administration of drugs in the form of a controlled release formulationis especially preferred in cases in which the drug has (i) a narrowtherapeutic index (i.e., the difference between the plasma concentrationleading to harmful side effects or toxic reactions and the plasmaconcentration leading to a therapeutic effect is small; in general, thetherapeutic index, TI, is defined as the ratio of median lethal dose(LD50) to median effective dose (ED50)); (ii) a narrow absorption windowin the gastrointestinal tract; or (iii) a very short biologicalhalf-life so that frequent dosing during a day is required in order tosustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the drug in question. Controlled release may be obtainedby appropriate selection of various formulation parameters andingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the drug is formulated with appropriateexcipients into a pharmaceutical composition that, upon administration,releases the drug in a controlled manner (single or multiple unit tabletor capsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the composition ofthe invention in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, microcrystalline cellulose, starches includingpotato starch, calcium carbonate, sodium chloride, calcium phosphate,calcium sulfate, or sodium phosphate); granulating and disintegratingagents (e.g., cellulose derivatives including microcrystallinecellulose, starches including potato starch, croscarmellose sodium,alginates, or alginic acid); binding agents (e.g., acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., stearic acid, silicas, or talc).Other pharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). A time delay material suchas, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology.

Drugs may be mixed together in the tablet, or may be partitioned. Forexample, a first drug is contained on the inside of the tablet, and asecond drug is on the outside, such that a substantial portion of thesecond drug is released prior to the release of the first drug.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, microcrystalline cellulose,calcium carbonate, calcium phosphate or kaolin), or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, liquid paraffin, or olive oil. Powders andgranulates may be prepared using the ingredients mentioned above undertablets and capsules in a conventional manner.

Controlled release compositions for oral use may, e.g., be constructedto release the active drug by controlling the dissolution and/or thediffusion of the active drug substance.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of drugs, or by incorporating the drug into an appropriatematrix. A controlled release coating may include one or more of thecoating substances mentioned above and/or, e.g., shellac, beeswax,glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated metylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more of the drugs ofthe claimed combinations may also be in the form of a buoyant tablet orcapsule (i.e., a tablet or capsule that, upon oral administration,floats on top of the gastric content for a certain period of time). Abuoyant tablet formulation of the drug(s) can be prepared by granulatinga mixture of the drug(s) with excipients and 20-75% w/w ofhydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropyl-methylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of anaqueous suspension by addition of water are convenient dosage forms fororal administration. Formulation as a suspension provides the activeingredient in a mixture with a dispersing or wetting agent, suspendingagent, and one or more preservatives. Suitable suspending agents are,for example, sodium carboxymethylcellulose, methylcellulose, sodiumalginate, and the like.

Parenteral Compositions

The pharmaceutical composition may also be administered parenterally byinjection, infusion or implantation (intravenous, intramuscular,subcutaneous, or the like) in dosage forms, formulations, or viasuitable delivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants. The formulation andpreparation of such compositions are well known to those skilled in theart of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active drug(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active drug(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. The composition may include suspending, solubilizing,stabilizing, pH-adjusting agents, and/or dispersing agents.

The pharmaceutical compositions according to the invention may be in theform suitable for sterile injection. To prepare such a composition, thesuitable active drug(s) are dissolved or suspended in a parenterallyacceptable liquid vehicle. Among acceptable vehicles and solvents thatmay be employed are water, water adjusted to a suitable pH by additionof an appropriate amount of hydrochloric acid, sodium hydroxide or asuitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodiumchloride solution. The aqueous formulation may also contain one or morepreservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). Incases where one of the drugs is only sparingly or slightly soluble inwater, a dissolution enhancing or solubilizing agent can be added, orthe solvent may include 10-60% w/w of propylene glycol or the like.

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the activedrug(s) may be incorporated in biocompatible carriers, liposomes,nanoparticles, implants, or infusion devices. Materials for use in thepreparation of microspheres and/or microcapsules are, e.g.,biodegradable/bioerodible polymers such as polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine). Biocompatible carriersthat may be used when formulating a controlled release parenteralformulation are carbohydrates (e.g., dextrans), proteins (e.g.,albumin), lipoproteins, or antibodies. Materials for use in implants canbe non-biodegradable (e.g., polydimethyl siloxane) or biodegradable(e.g., poly(caprolactone), poly(glycolic acid) or poly(ortho esters)).

Alternative Routes

Although less preferred and less convenient, other administrationroutes, and therefore other formulations, may be contemplated. In thisregard, for rectal application, suitable dosage forms for a compositioninclude suppositories (emulsion or suspension type), and rectal gelatincapsules (solutions or suspensions). In a typical suppositoryformulation, the active drug(s) are combined with an appropriatepharmaceutically acceptable suppository base such as cocoa butter,esterified fatty acids, glycerinated gelatin, and various water-solubleor dispersible bases like polyethylene glycols. Various additives,enhancers, or surfactants may be incorporated.

The pharmaceutical compositions may also be administered topically onthe skin for percutaneous absorption in dosage forms or formulationscontaining conventionally non-toxic pharmaceutical acceptable carriersand excipients including microspheres and liposomes. The formulationsinclude creams, ointments, lotions, liniments, gels, hydrogels,solutions, suspensions, sticks, sprays, pastes, plasters, and otherkinds of transdermal drug delivery systems. The pharmaceuticallyacceptable carriers or excipients may include emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

The preservatives, humectants, penetration enhancers may be parabens,such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride,glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topicaladministration on the skin may also be used in connection with topicaladministration onto or close to the part of the body that is to betreated. The compositions may be adapted for direct application or forapplication by means of special drug delivery devices such as dressingsor alternatively plasters, pads, sponges, strips, or other forms ofsuitable flexible material.

Dosages and Duration of the Treatment

It will be appreciated that the drugs of the combination may beadministered concomitantly, either in the same or differentpharmaceutical formulation or sequentially. If there is sequentialadministration, the delay in administering the second (or additional)active ingredient should not be such as to lose the benefit of theefficacious effect of the combination of the active ingredients. Aminimum requirement for a combination according to this description isthat the combination should be intended for combined use with thebenefit of the efficacious effect of the combination of the activeingredients. The intended use of a combination can be inferred byfacilities, provisions, adaptations and/or other means to help using thecombination according to the invention.

Therapeutically effective amounts of the drugs in a combination of thisinvention include, e.g., amounts that are effective for reducingParkinson's disease symptoms, halting or slowing the progression of thedisease once it has become clinically manifest, or prevention orreduction of the risk of developing the disease.

Although the active drugs of the present invention may be administeredin divided doses, for example two or three times daily, a single dailydose of each drug in the combination is preferred, with a single dailydose of all drugs in a single pharmaceutical composition (unit dosageform) being most preferred.

Administration can be one to several times daily for several days toseveral years, and may even be for the life of the patient. Chronic orat least periodically repeated long-term administration is indicated inmost cases.

The term “unit dosage form” refers to physically discrete units (such ascapsules, tablets, or loaded syringe cylinders) suitable as unitarydosages for human subjects, each unit containing a predeterminedquantity of active material or materials calculated to produce thedesired therapeutic effect, in association with the requiredpharmaceutical carrier.

The amount of each drug in a preferred unit dosage composition dependsupon several factors including the administration method, the bodyweight and the age of the patient, the stage of the disease, the risk ofpotential side effects considering the general health status of theperson to be treated. Additionally, pharmacogenomic (the effect ofgenotype on the pharmacokinetic, pharmacodynamic or efficacy profile ofa therapeutic) information about a particular patient may affect thedosage used.

Except when responding to especially impairing cases, where higherdosages may be required, the preferred dosage of each drug in thecombination will usually lie within the range of doses not above thedosage usually prescribed for long-term maintenance treatment or provento be safe in phase 3 clinical studies.

One remarkable advantage of the invention is that each compound may beused at low doses in a combination therapy, while producing, incombination, a substantial clinical benefit to the patient. Thecombination therapy may indeed be effective at doses where the compoundshave individually low or no effect. Accordingly, a particular advantageof the invention lies in the ability to use sub-optimal doses of eachcompound, i.e., doses which are lower than therapeutic doses usuallyprescribed, preferably ½ of therapeutic doses, more preferably ⅓, ¼, ⅕,or even more preferably 1/10 of therapeutic doses. In particularexamples, doses as low as 1/20, 1/30, 1/50, 1/100, or even lower, oftherapeutic doses are used.

As mentioned above, this is likely due to a simultaneous activity ofcompositions of the invention on several targets involved in thealpha-synuclein aggregation network. In a particular embodiment suchconjunction of molecular effects can further lead to synergisticcombinations. Synergy may be assessed by methods known well known bythose skilled in the art. For instance, synergy can be characterized byusing a two way ANOVA to determine whether the interaction between eachdrugs is significant or not (i.e. synergy, [32]), or by calculating acombinatory index from the dose effect curves of each of the compoundsalone and of their combinations [33, 34].

At such sub-therapeutic dosages, the compounds would exhibit no sideeffect, while the combination(s) according to the invention are fullyeffective in treating parkinsonism, more preferably PD.

A preferred dosage corresponds to amounts from 1% up to 50% of thoseusually prescribed for long-term maintenance treatment.

The most preferred dosage may correspond to amounts from 1% up to 10% ofthose usually prescribed for long-term maintenance treatment.

Specific examples of dosages of drugs (Quantity equivalent to activemolecule) for use in the invention are provided below:

-   -   acamprosate: 1000 mg or less per day, preferably less than 500        mg per day, preferably less than 400 mg per day, more preferably        less than 200 mg per day, more preferably less than 50 mg per        day, or even less that than 10 mg per day; even more preferably        from about 0.1 to 1000 mg per day, furthermore preferably        between 0.5 mg and 100 mg, typically 0.8 mg per day, 2 mg per        day, 20 mg per day, 40 mg per day, or 80 mg per day, such        dosages being particularly suitable for oral administration,    -   baclofen: 150 mg or less per day, preferably less than 100 mg        per day, more preferably less than 50 mg per day, more        preferably less than 30 mg per day, even more preferably between        0.01 mg and 30 mg per day, typically 12 mg per day, 24 mg per        day, 30 mg per day, such dosages being particularly suitable for        oral administration, such dosages being particularly suitable        for oral administration,    -   cinacalcet: 150 mg or less per day, preferably less than 100 mg        per day, preferably less than 50 mg per day, more preferably        less than 36 mg per day, and even more preferably between 0.01        and 25 mg per day, such dosages being particularly suitable for        oral administration,    -   mexiletine: 120 mg or less per day, preferably less than 60 mg        per day, more preferably less than 30 mg per day, more        preferably less than 15 mg per day, even more preferably between        6 and 15 mg per day, such dosages being particularly suitable        for oral administration,    -   torasemide: 4 mg or less per day, preferably less than 2 mg per        day, more preferably less than 1 mg per day, more preferably        less than 0.5 mg per day, and even more preferably between 0.05        and 0.5 mg per day, such dosages being particularly suitable for        oral administration,    -   sulfisoxazole: 800 mg or less per day, preferably less than 400        mg, more preferably less than 200 mg per day, more preferably        less than 100 mg per day, even more preferably less than 20 mg        per day, such dosages being particularly suitable for oral        administration,    -   tadalafil: 20 mg or less per day, preferably less than 10 mg per        day, more preferably less than 4 mg per day, more preferably        less than 2.5 mg per day, and even more preferably between 0.025        and 2.5 mg per day, such dosages being particularly suitable for        oral administration,    -   levodopa: 1.5 g or less per day, preferably less than 750 mg per        day, more preferably less than 375 mg per day, even more        preferably less than 100 mg per day, such dosages being        particularly suitable for oral administration,    -   rasagiline: 0.5 mg or less per day, preferably less than 0.25 mg        per day, more preferably less than 0.1 mg per day, more        preferably less than 0.05 mg per day, even more preferably        between 0.005 and 0.05 mg per day, such dosages being        particularly suitable for oral administration,    -   selegiline: 5 mg or less per day, preferably less than 2.5 mg        per day, more preferably less than 1 mg per day, more preferably        less than 0.5 mg per day, even more preferably between 0.05 and        0.1 mg per day, such dosages being particularly suitable for        oral administration.

In a particularly preferred embodiment, combinatorial therapies of theinvention comprise administering between 0.4 mg and 50 mg of acamprosateand 6 mg to 15 mg baclofen, twice daily.

In an embodiment, combinatorial therapies the invention compriseadministering 0.4 mg acamprosate and 6 mg baclofen, twice daily.

In a preferred embodiment, combinatorial therapies of the invention ofthe invention comprise administering 1 mg acamprosate and 15 mgbaclofen, twice daily.

In yet another preferred embodiment, combinatorial therapies of theinvention comprise administering 10 mg acamprosate and 6 mg baclofen,twice daily.

In another preferred embodiment, combinatorial therapies of theinvention comprise administering 20 mg acamprosate and 12 mg baclofen,twice daily.

In still another embodiment, combinatorial therapies of the inventioncomprise administering 40 mg acamprosate and 12 mg baclofen, twicedaily.

In still another embodiment, combinatorial therapies of the inventioncomprise administering 40 mg acamprosate and 30 mg baclofen, twicedaily.

In another particular embodiment, besides comprising administering oneof the above baclofen-acamprosate regimen, therapies of the inventionalso comprise administering levodopa or melevodopa either at their usualdose and regimen (i.e. as an add-on therapy) or even at a lower dose,from 1% up to 50% of those usually prescribed for the treatment ofParkinson's disease.

In a further particular embodiment, besides comprising administering oneof the above baclofen-acamprosate regimen, therapies of the inventionalso comprise administering rasagiline or selegiline either at theirusual dose and regimen (i.e. as an add-on therapy) or even at a lowerdose, from 1% up to 50% of those usually prescribed for the treatment ofPD.

When the composition comprises baclofen and acamprosate, these twocompounds may be used in different ratios, e.g., at a weight ratiobaclofen/acamprosate comprised between from 0.05 to 1000 (W:W),preferably between 0.05 to 100 (W:W), more preferably between 0.05 to62.5 (W:W), even more between preferably 0.3 to 15 (W:W). Typically suchweight ratio baclofen/acamprosate is 0.3, 0.6, 0.75, 1.2, 3.1, 12, 15,25 or 62.5.

It will be understood that the amount of the drug or the drugcombination actually administered will be determined by a physician, inthe light of the relevant circumstances including the condition orconditions to be treated, the exact composition to be administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the chosen route of administration. Therefore,the above dosage ranges are intended to provide general guidance andsupport for the teachings herein, but are not intended to limit thescope of the invention.

The following examples are given for purposes of illustration and not byway of limitation.

EXAMPLES

All animal procedures have been conducted in compliance to the NationalInstitute of Health (NIH) guidelines for the care and use of laboratoryanimals, and approved by the National Animal Experiment Board.

A—Prevention of Glutamate Toxicity on Neuronal Cells

Glutamate toxicity is involved in the pathogenesis of Parkinson'sdisease. In this set of experiment, candidate compounds have been testedfor their ability to prevent or reduce the toxic effects of glutamatetoxicity on neuronal cells. The drugs are first tested individually,followed by assays of their combinatorial action.

Neuronal Cell Preparation

The efficacy of drug combinations of the invention is assessed onprimary cortical neuron cells.

Rat cortical neurons were cultured as described by Singer et al. [35].Briefly pregnant female rats of 15 days gestation were killed bycervical dislocation (Rats Wistar) and the foetuses were removed fromthe uterus. The cortex was removed and placed in ice-cold medium ofLeibovitz (L15) containing 2% of Penicillin 10.000 U/ml and Streptomycin10 mg/ml and 1% of bovine serum albumin (BSA). Cortices were dissociatedby trypsin for 20 min at 37° C. (0.05%). The reaction was stopped by theaddition of Dulbecco's modified Eagle's medium (DMEM) containing DNase1grade II and 10% of foetal calf serum (FCS). Cells were thenmechanically dissociated by 3 serial passages through a 10 ml pipetteand centrifuged at 515 g for 10 min at +4° C. The supernatant wasdiscarded and the pellet of cells was re-suspended in a defined culturemedium consisting of Neurobasal supplemented with B27 (2%), L-glutamine(0.2 mM), 2% of PS solution and 10 ng/ml of BDNF. Viable cells werecounted in a Neubauer cytometer using the trypan blue exclusion test.The cells were seeded at a density of 30 000 cells/well in 96well-plates (wells were pre-coated with poly-L-lysine (10 μg/ml)) andwere cultured at +37° C. in a humidified air (95%)/CO2 (5%) atmosphere.

Glutamate Toxicity Assays

The neuroprotective effect of compounds is assessed by quantification ofthe neurite network (Neurofilament immunostaining (NF) whichspecifically reveals the glutamatergic neurons).

After 12 days of neuron culture, drugs of the candidate combinations aresolved in culture medium (+0.1% DMSO). Candidate combinations are thenpre-incubated with neurons for 1 hour before the glutamate injury. Onehour after incubation with, glutamate is added for 20 min, to a finalconcentration of 40 μM, in presence of candidate combinations, in orderto avoid further drug dilutions. At the end of the incubation, medium ischanged with medium with candidate combination but without glutamate.The culture is fixed 24 hours after glutamate injury. MK801(dizocilpinehydrogen maleate, 77086-22-7-20 μM) is used as a positivecontrol.

After permeabilization with saponin (Sigma), cells are blocked for 2 hwith PBS containing 10% goat serum, then the cells are incubated withmouse monoclonal primary antibody against Neurofilament antibody (NF,Sigma). This antibody is revealed with Alexa Fluor 488 goat anti-mouseIgG.

Nuclei of cells are labeled by a fluorescent marker (Hoechst solution,SIGMA), and neurite network quantified. Six wells per condition are usedto assess neuronal survival in 3 different cultures.

Results

All of the tested drug combinations give a protective effect againstglutamate toxicity for cortical neuronal cells. Results are shown inTable 2 below.

As exemplified in FIGS. 1 to 3, combinations of the invention stronglyprotect neurons from glutamate toxicity under experimental conditionsdescribed above. It is noteworthy that an effective protection isnoticed using drug concentrations at which drugs used alone have nosignificant or lower protective effect.

Indeed, as exemplified in FIG. 1, mexiletine-cinacalcet combinationefficiently protects neuronal cells from glutamate toxicity, whereas noprotection is afforded by the single drugs. Baclofen-acamprosate (FIG.3) combination gives a protective effect against glutamate toxicity forcortical neuronal cells. Combination of baclofen and acamprosate inducesan improvement of more than 200% compared to acamprosate alone and ofmore than 47% compared to baclofen used alone.

TABLE 2 Neuroprotective effect against Drug Combination glutamatetoxicity baclofen and torasemide + baclofen-acamprosate-torasemide +mexiletine and cinacalcet + sulfisoxazole and torasemide + baclofen andacamprosate + acamprosate and cinacalcet + baclofen and cinacalcet +B—Protective Effect Against Ischemia/Hypdxia Induced Neuronal CellDeath.Rat Neuronal Cortical Cells Preparation

Cells are prepared as previously.

Oxygen and Glucose Deprivation Assays (In Vitro Model of Ischemia)

The neuroprotective effect of compounds is assessed by quantification ofthe neurite network using MAP2 antibody. Riluzole, a neuroprotectivedrug, (Riluteck®, 5 μM) is used as positive control.

After 10 days of neuron culture, candidate drugs are solved in culturemedium (+0.1% DMSO) and then pre-incubated with neurons for 1 hourbefore the oxygen and glucose deprivation. One hour after candidate drugincubation, the medium is removed and fresh medium without glucose isadded. This medium is composed by DMEM without glucose (Invitrogen)supplemented with 2% B27, 0.2 mM L-glutamine, 1% PS solution, 10 ng/mlof BDNF. The cells are transferred into an anaerobic incubator with 95%N₂ and 5% CO₂ at 37° C.

After 2 hours, 25 mM of D-Glucose will be added in culture medium andcells are transferred in classic incubator with 95% air/5% CO₂ at 37° C.After 24 hours of oxygen glucose reperfusion, cells are fixed by a coldsolution of alcohol/acetic acid during 5 minutes.

After permeabilization with saponin (Sigma), cells are blocked for 2hours with PBS containing 10% goat serum, then the cells are incubatedwith mouse monoclonal primary antibody against MAP2 (MAP2, Sigma). Theseantibodies are revealed with Alexa Fluor 488 goat anti-mouse IgG(Molecular probe).

Nuclei of cells are labelled by a fluorescent marker (Hoechst solution,SIGMA). Six wells per condition are used to assess neuronal survival in3 different cultures.

For each condition 2×10 pictures per well are taken and analyzed usingInCell Analyzer™ 1000 (GE Healthcare) with 20× magnification.

Results

As shown in Table 3 below, all of the claimed drug combinations give aprotective effect against ischemia/hypoxia induced cell death forcortical neuronal cells.

TABLE 3 Protective effect against Drug Combination ischemia/hypoxiabaclofen and torasemide + baclofen - acamprosate - torasemide +mexiletine and cinacalcet + sulfisoxazole and torasemide + baclofen andacamprosate + acamprosate and cinacalcet + baclofen and cinacalcet +

FIGS. 4-6 further show that the combination treatments of the inventionstrongly protect neurons from oxygen and glucose deprivation. As shownin FIGS. 4-6, an effective protection is observed using drugconcentrations at which drugs, alone, have no significant protectiveeffect. For example, a significant protective effect of baclofen (80nM)/acamprosate (0.32 nM) combination or cinacalcet (64 pM)/mexiletine(25.6 pM) combination or torasemide (80 nM)/sulfisoxazole (1.36 nM), inischemia is observed, while no significant protection is obtained whenbaclofen, acamprosate, cinacalcet, mexiletine, torasemide andsulfisoxazole are used alone, at the same concentrations.

These results therefore demonstrate a potent and synergistic effect ofthe combination therapies on oxidative stress and mitochondrialdysfunction or apoptosis which are underlie under ischemic conditions aswell as PD.

C— Neuro-Protective Effect of Drugs Against 6-OHDA Injury onDopaminergic Neurons

6-hydroxydopamine (6-OHDA) is a neurotoxic drug which selectivelyinitiates neuronal degeneration of dopaminergic neurons by generatingreactive oxygen species and mitochondrial respiratory dysfunction whichare thought to mirror events occurring in parkinsonian brain. 6-OHDAtoxicity is commonly used in vitro and in vivo to study Parkinsonism.

Culture of Mesencephalic Dopaminergic Neurons

Rat dopaminergic neurons were cultured as described by Schinelli et al.[36]. Pregnant female rats of 15 days gestation were killed by cervicaldislocation (Rats Wistar; Janvier) and the foetuses removed from theuterus. The embryonic midbrains were removed and placed in ice-coldmedium of Leibovitz (L15; PanBiotech) containing 2% ofPenicillin-Streptomycin (PS; PanBiotech) and 1% of bovine serum albumin(BSA; PanBiotech). Only the ventral portions of the mesencephalicflexure were used for the cell preparations as this is the region of thedeveloping brain rich in dopaminergic neurons. The midbrains weredissociated by trypsinisation for 20 min at 37° C. (Trypsin EDTA 1×;PanBiotech). The reaction is stopped by the addition of Dulbecco'smodified Eagle's medium (DMEM; PanBiotech) containing DNAase I grade II(0.1 mg/ml; PanBiotech) and 10% of foetal calf serum (FCS; Invitrogen).Cells were then mechanically dissociated by 3 passages through a 10 mlpipette and centrifuged at 180×g for 10 min at +4° C. on a layer of BSA(3.5%) in L15 medium. The supernatant was discarded and the cells ofpellet were re-suspended in a defined culture medium consisting ofNeurobasal (Invitrogen) supplemented with B27 (2%; Invitrogen),L-glutamine (2 mM; PanBiotech) and 2% of PS solution and 10 ng/ml ofBrain-derived neurotrophic factor (BDNF, PanBiotech) and 1 ng/ml ofGlial-Derived Neurotrophic Factor (GDNF, PanBiotech). Viable cells werecounted in a Neubauer cytometer using the trypan blue exclusion test.The cells were seeded at a density of 40 000 cells/well in 96well-plates (pre-coated with poly-L-lysine (Greiner)) and are culturedat 37° C. in a humidified air (95%)/CO2 (5%) atmosphere. Half of themedium was changed every 2 days with fresh medium. Five to six percentof the neuronal cell population were dopaminergic neurons.

6-OHDA and Test Compounds Exposure

On day 6 of culture, the medium was removed and fresh medium was added,without or with 6-OHDA at the following concentrations: 20 μM during 48hours diluted in control medium. Test compounds were pre-incubated for 1h before the 6-OHDA application during 48 hours.

End Point Evaluation: Measure of Total Number of Tyrosine Hydroxylase(TH) Positive Neurons

After 48 hours of intoxication with 6-OHDA, cells were fixed by asolution of 4% paraformaldehyde (Sigma) in PBS, pH=7.3 for 20 min atroom temperature. The cells were washed again twice in PBS, and thenpermeabilized and non-specific sites were blocked with a solution of PBScontaining 0.1% of saponin (Sigma) and 1% FCS for 15 min at roomtemperature. Then, cells were incubated with Monoclonal Anti-TyrosineHydroxylase antibody produced in mouse (TH, Sigma) at dilution of 1/1000in PBS containing 1% FCS, 0.1% saponin, for 2 hours at room temperature.These antibodies were revealed with Alexa Fluor 488 goat anti-mouse IgG(Molecular Probes) at the dilution 1/800 in PBS containing 1% FCS, 0.1%saponin, for 1 hour at room temperature.

For each condition, 2×10 pictures (representing ˜80% of total well area)per well were taken using InCell Analyzer™ 1000 (GE Healthcare) with 10×magnification. All images were taken in the same conditions. Analysis ofthe number of TH positive neurons were done using Developer software (GEHealthcare).

Data are expressed in percentage of control conditions (no intoxication,no 6-OHDA=100%) in order to express the 6-OHDA injury. All values areexpressed as mean+/−SEM (s.e. mean) of the 3 cultures (n=6 wells percondition per culture). Statistical analyses consist in an ANOVAfollowed by the Dunnett's and PLSD Fisher's tests when it was allowedusing Statview software version 5.0.

Results

A neuroprotective effect is observed for combinations of the inventionin TH neurons survival test after 48 hour 6-OHDA injury on dopaminergicneurons.

A 48 h 6-OHDA (20 μM) incubation with mesencephalic neurons produced asignificant intoxication of dopaminergic neurons (around −33% of THneurons) in all the experiments (control, FIGS. 7-9,12-14).

BDNF was used as a positive control. One hour of BDNF pre-treatment at1.85 nM significantly protected the dopaminergic neurons from this6-OHDA injury.

As shown in table 4 below, all of the claimed drug combinations give aprotective effect against 6-OHDA injury in dopaminergic neuronal cells.

TABLE 4 Protective effect against 6-OHDA Drug Combination injury indopaminergic neuronal cells baclofen and torasemide +baclofen-acamprosate-torasemide + mexiletine and cinacalcet +sulfisoxazole and torasemide + baclofen and acamprosate + acamprosateand cinacalcet + baclofen and cinacalcet + cinacalcet and tadalafil +

As shown in FIGS. 7-9 and 12-14, baclofen-acamprosate,baclofen-torasemide, mexiletine-cinacalcet, acamprosate-cinacalcet,cinacalcet-tadalafil successfully protect dopaminergic neurons from6-OHDA toxicity.

Notably, baclofen-acamprosate combination is shown effective inprotecting, in vitro, dopaminergic neurons for a large range ofconcentrations of baclofen (from 16 nM to 400 nM) and acamprosate (from4 pM to 1600 pM).

Moreover inventors have been able to draw dose-effect curves for eachbaclofen, acamprosate, cinacalcet and tadalafil (not shown) whichfurther allow to determine the combinatory index (CI) of the combinationof these drugs according to Loewe [33, 34]. A combinatory index below 1characterizes synergy between drugs within a given combination.

As illustrated in FIG. 12, the baclofen-acamprosate mix displaying asynergistic activity in the above glutamate toxicity and ischemia invitro models, is also found as having a synergistic activity in the6-OHDA toxicity model. For example, baclofen at 32 nM when combined withacamprosate at either 4 pM or 10 pM show a synergistic protectiveactivity (S, FIG. 12) on 6-OHDA intoxicated dopaminergic neuronal cells,with a CI=0.4 or 0.5 respectively.

Tested combinations of acamprosate-cinacalcet at different drugconcentrations were also found to have a synergistic protective effectagainst 6-OHDA induced toxicity. Their neuroprotective effect isparticularly important, especially when considering the very lowconcentrations of the drugs, as illustrated in FIG. 13 and table 5.Combinatory indexes related to the different tested compositions are, byfar, below the limit of 1 which denotes a strong synergistic protectiveeffect of the mix acamprosate-cinacalcet on dopaminergic neurons (table5).

TABLE 5 Mix acamprosate-cinacalcet Protection Combinatory AcamprosateCinacalcet (% of control) index Dose 1 2 pM 1.6 nM +71% 0.00016 Dose 2 2pM 8 nM +38% 0.00848 Dose 3 2 pM 40 nM +17% 0.344 Dose 4 10 pM  1.6 nM+22% 0.138 Dose 5 4 pM 1.6 nM +41% 0.0024 Dose 6 4 pM 40 nM +23% 0.276

Tested combinations of cinacalcet-tadalafil were also found efficient inprotecting dopaminergic neurons from 6-OHDA toxicity. This protectiveeffect against 6-OHDA induced toxicity has been found synergistic forvarious doses of individual drugs. Their neuroprotective effect isillustrated in FIG. 14. Combinatory indexes related to the differenttested compositions are below the limit of 1 which denotes a strongsynergistic protective effect of the mix cinacalcet-tadalafil ondopaminergic neurons (table 6).

TABLE 6 Mix cinacalcet-tadalafil Protection Combinatory cinacalcettadalafil (% of control) index Dose 1 1.6 nM 1 nM +25% 0.676 Dose 2 1.6nM 10 nM +40% 0.781 Dose 3 8 nM 1 nM +25% 0.651 Dose 4 40 nM 0.5 nM +30%0.336 Dose 5 40 nM 1 nM +28% 0.692 Dose 6 40 nM 10 nM +30% N/A Dose 7 8nM 10 nM +35% N/A N/A: not availableD—Effects on Dopaminergic Neuronal Loss In Vivo and on Motor Symptoms

1) Improvement of Akinesia In Vivo

Animal Husbandry and Surgical Procedure

Wistar rats (5 weeks) were used after an acclimatization period of atleast 5 days. Surgery was performed under ketamine (50 mg/kg) andxylazine (10 mg/kg). Animals received an unilateral injection of 12 μgof 6-OHDA (sigma Aldrich) dissolved in 6 μl of 0.9% sterile NaClcontaining 0.1% ascorbic acid (to protect 6-OHDA from oxidation), at theflow rate of 1 μl/min, in the left substantia nigra pars compacta. Thestereotaxic coordinates of the injection site will be:anteroposterior+2.2 mm, lateral 2.0 mm, dorsoventral+3 mm with theincisor bar at +5.0 mm above the interaural plane, according to the ratstereotaxic atlas by De Groot (1959) [37].

Drug(s) Treatment

The first administration of treatment or vehicle has been performed theday before the stereotaxic injection of 6-OHDA (for the lesion groups)or vehicle and all along the 15 days preceding the behavioral tests.Rats of the reference treatment group were administered with acombination of L-DOPA (8 mg/kg) and benserazide (a peripheral DPAdecarboxylase inhibitor, 12.5 mg/kg). During the study and for eachanimal, the volume of per os administrations was determined on the basisof the mean body weight of the animals of the corresponding group. Bodyweights will be determined twice a week and the volume of administrationwill thus be adjusted consequently.

Vehicles and compounds have been administered twice a day (i.e. bid: bisin die) through the oral route, in the morning and in the afternoon;eight hours (+/−30 min) separated the two administrations around 9:30 AMand around 17:30 PM.

On the day of behavioral tests, the drugs have been administered around1 hour for L-DOPA treated group and around 2 hour (+/−15 min) for thedrug combinations before the behavioral test, for each animal.

Behavioral Testing

Each test was performed before surgery (2 or 3 days before) to determinethe basal level value. Assessment of behavioral functions is performed15 days after surgical injection of 6-OHDA using the two differenttests.

Initiation Time Test (ITT):

The animal was hold by a trained technician in front of a plane surface.Only one of the two forelimbs was left free to move. The time that wasnecessary to initiate the movement toward the plane surface has beenrecorded using 180 sec as break-off point [38].

Stepping Test (ST):

The rat was hold by the experimenter and only one of the two forelimbshave been left free to move above a plane surface. The other hand fixedthe forelimb not to be monitored with one paw touching the table. Theanimal has then been moved slowly backward or forward (5 sec for 0.9 m)by the experimenter. The number of adjusting steps was counted for theright paw [38].

Cylinder Test (CT):

The rat was placed in plexiglass cylinder and, immediately after,videotaped for 15 min to examine the symmetry/asymmetry of theirforepaws use during explorative behavior in this new environment. Thenumbers of contacts made on the cylinder wall during this period withthe ipsilateral paw, the contralateral paw, and with both paws (doublecontacts) will be determined and expressed as a percentage of the totalnumber of contacts [39, 40].

Results

In vivo assays were carried out with drug combinations of the invention.Tested drug combinations of the invention induced a significantimprovement either in the initiation time test or stepping test (table7).

TABLE 7 Protective effect against Drug Combination 6-OHDA inducedakinesia baclofen and torasemide + baclofen - acamprosate - torasemide +mexiletine and cinacalcet + sulfisoxazole and torasemide + baclofen andacamprosate + acamprosate and cinacalcet + baclofen and cinacalcet +cinacalcet and tadalafil +

As exemplified in FIGS. 10 and 11, drug combinations of the inventionstrongly protect rats from 6-OHDA stereotaxic lesions. Noteworthy,treatment with baclofen-acamprosate combination results in an almostfull alleviation of akinesia in the stepping test and in the initiationtest, in a dose dependent manner.

This effect on the combinations of the invention on akinesia was alsoassessed in a different test (cylinder test) also used to evaluateakinesia. Results are illustrated in FIG. 15 show that thebaclofen-acamprosate (3.75 mg/kg bid and 0.25 mg/kg bid, respectively)combination alleviates the lowering of double contacts on the cylinderinduced by 6-OHDA intoxication, which shows an improvement of the ratspontaneous motor behavior.

Several doses and ratios of the drugs have been tested for thebaclofen-acamprosate combination. Results gathered in table 8 belowconfirm that a large range of doses or ratios of baclofen andacamprosate are efficient in counteracting akinesia induced by thestereotaxic injection of 6-OHDA.

TABLE 8 Protective effect against Baclofen-acamprosate (BCL-ACP)treatment 6-OHDA induced akinesia* dose 1: 0.6 mg/kg bid and 0.04 mg/kgbid + dose 2: 1.5 mg/kg bid and 0.1 mg/kg bid + dose 3: 3.75 mg/kg bidand 0.25 mg/kg bid + dose 4: 125 μg/kg bid and 2 μg/kg bid + dose 5: 125μg/kg bid and 40 μg/kg bid + dose 6: 0.5 mg/kg bid and 8 μg/kg bid +dose 7: 0.5 mg/kg bid and 20 μg/kg bid + *in at least one of ITT, CT orST

2) Improvement of Akinesia In Vivo is Related to a Reduced Neuronal Lossin Treated Animals

Tissue Preparation for Macrohistology

All animals were killed by lethal injection of pentobarbital. The brainswere quickly removed, then frozen in dry ice and stored at −80° C.

Striatum slices preparation: Coronal (10 μm thick) tissue sections atthe level of striatum were cut at −20° C. with a cryostat (HM560). Threeserial striatal sections per animal were then mounted on SuperFrost Plusglass slides (Fisher Scientific) and stored at −80° C. untilhistological analysis.

Subtantia nigra pars compacta (SN) slices preparation: slices aredistributed in three sets of sections covering the whole SN(coordinates: −4.8, −5.3 and −5.8 mm AP from bregma). SN slices, afterrinses, were immersed in 0.3% H₂O₂, pre-incubated for 30 min in PBScontaining 5% BSA, and then incubated overnight in the same solutioncontaining a monoclonal Anti-Tyrosine Hydroxylase antibody (TH, Sigma)at the dilution of 1/1000. Section are subsequently incubated for twohours with a dye labelled anti-mouse secondary antibody.

Labelling of Dopaminergic Terminals Neurons in Striatum with[³H]-Mazindol Staining

The loss of DA terminals in the striatum was assessed as an index of theextent of the dopaminergic denervation by analysis of [³H]-mazindolbinding to dopamine uptake sites [40]. Indeed, mazindol is a blocker ofdopamine reuptake transporter which is commonly used to labeldopaminergic neurons in experimental biology.

Briefly, striatal section were air dried (with a laboratory air dryer)and rinsed for 5 min in 50 mM Tris buffer with 120 mM NaCl and 5 mM KCl.They were then incubated for 40 min with 15 nM [³H]-mazindol (NEN,DuPont; specific activity, 17 Ci/mM) in 50 mM Tris buffer containing 300mM NaCl and 5 mM KCl added with 0.3 mM desipramine to block thenoradrenalin uptake sites. Sections were rinsed twice for 3 min in theTris incubation buffer and for 10 s in distilled water and were airdried. [³H]-sensitive photographic fit (Kodal BioMax MS Film, Sigma) wasexposed to the slices in x-ray cassettes and exposed at room temperaturefor 3 weeks. The levels of [³H]-mazindol labelling was quantified bydigitized image analysis from the film autoradiograms using as BIOCOManalysis system (Densirag, BIOCOM). Grey levels were converted tooptical densities (ODs) using external standards (calibrated densitystep tablet, Kodak). The mean OD value was determined from threesections per animal after subtracting the background signal measured oneach section by scanning an area of the corpus callosum that is known tolack DA terminals.

Quantification of TH Positive Neuron in SN

TH positive neurons were counted in a set of the three sections centeredon the track of the 6-OHDA injection needle in the SN, covering almostall the SN. The specificity of DA neuron damage, global cells in the SNare counted on toluidine blue labelled sections of the same region. Cellcounted is done using a microscope connected to a computer imageanalysis system by a CDD camera.

Results

The 6-OHDA injection in the left SN results in the decrease of thedensity of both the DA cellular bodies in the SN and the DA terminalsdensity in the left side of the striatum (FIG. 16). L-DOPA, which isrecognized as an only symptomatic treatment of PD, failed to inhibitthis 6-OHDA induced loss of neurons. Administration of dose 3 ofbaclofen-acamprosate combination (3.75 mg/kg bid and 0.25 mg/kg bid,respectively) induced a statistically significant elevation of DAneurons in the SN and also in the striatum when compared withnon-treated animals (FIG. 16). Hence baclofen-acamprosate combinationaffords an actual protection of DA neurons against degeneration inducedby 6-OHDA to the difference of L-DOPA, the currently most efficienttreatment though only symptomatic. Not only cellular bodies of DAneurons of SN but also nerve terminals in the striatum are protected,hence this protection seems to act also against an anterograde neuronaldegeneration.

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The invention claimed is:
 1. A method of treating Parkinson's disease ina subject in need thereof, comprising administering to said subject aneffective amount of tadalafil and cinacalcet and, optionally, a dopamineprecursor, a dopamine receptor agonist, an inhibitor of a dopaminemetabolizing enzyme, baclofen, mexiletine, sulfisoxazole and torasemide,or salt(s), prodrug(s) or sustained release formulation(s) thereof asthe sole active ingredients, wherein the subject has Parkinson'sdisease.
 2. The method of claim 1, said method comprising administeringto said subject a further compound selected from baclofen, mexiletine,sulfisoxazole and torasemide or salt(s), prodrug(s) or sustained releaseformulation(s) thereof.
 3. The method of claim 2, comprisingadministering to said subject at least one of the following combinationsof compounds: tadalafil, cinacalcet and mexiletine, or tadalafil,cinacalcet and baclofen, or salt(s), prodrug(s) or sustained releaseformulation(s) thereof.
 4. The method of claim 1, comprising furtheradministering to said subject at least one of: a dopamine precursor, adopamine receptor agonist, or an inhibitor of a dopamine metabolizingenzyme.
 5. The method of claim 4, wherein the dopamine precursor islevodopa or melevodopa or salts, prodrugs or sustained releaseformulation thereof; the dopamine receptor agonist is a compoundselected from the group consisting of talipexole, piribedil, rotigotine,bromocriptine, pergolide, cabergoline, lisuride, pramipexole, ropiniroleor apomorphine or salts, prodrugs or sustained release formulationsthereof; and the inhibitor of a dopamine metabolizing enzyme is acompound selected from the group consisting of carbidopa, benserazide,entacapone, tolcapone, selegiline and rasagiline, or salt(s), prodrug(s)or sustained release formulations thereof.
 6. The method of claim 5,comprising administering to said subject at least one of the followingcombinations of compounds: tadalafil, cinacalcet and levodopa,tadalafil, cinacalcet, mexiletine and levodopa, or tadalafil,cinacalcet, baclofen and levodopa, or salt(s), prodrug(s) or sustainedrelease formulation(s) thereof.
 7. The method of claim 6, furthercomprising administering to said subject at least one compound selectedfrom carbidopa, benserazide, entacapone, tolcapone, selegiline andrasagiline, or a salt, prodrug or sustained release formulation thereof.8. The method of claim 7, wherein said at least one compound iscarbidopa, or a salt, prodrug or sustained release formulation thereof.9. The method of claim 5, comprising administering to said subject atleast one of the following combinations of compounds: tadalafil,cinacalcet and selegiline or rasagiline, tadalafil, cinacalcet,mexiletine and selegiline or rasagiline, or tadalafil, cinacalcet,baclofen and selegiline or rasagiline, or salt(s), prodrug(s) orsustained release formulation(s) thereof.
 10. The method of claim 1,wherein the compounds are administered with a pharmaceuticallyacceptable carrier or excipient.
 11. The method of claim 1, wherein thecompounds are formulated or administered together, separately orsequentially.
 12. The method of claim 1, wherein the compounds areadministered repeatedly to the subject.
 13. The method of claim 1,wherein the compounds are administered orally.
 14. The method of claim1, wherein said administration protects dopaminergic neurons of thenigrostriatal system of the subject from degeneration or death.
 15. Themethod of claim 1, wherein said administration treats bradykinesia orakinesia in said subject.
 16. The method of claim 1, wherein saidsubject is also treated with deep brain stimulation of the subthalamicnucleus or of the globus pallidus interna.
 17. The method of claim 1,said method comprising administering tadalafil and cinacalcet orsalt(s), prodrug(s) or sustained release formulation(s) thereof as thesole active ingredients.
 18. A composition comprising tadalafil andcinacalcet and, optionally, a dopamine precursor, a dopamine receptoragonist, an inhibitor of a dopamine metabolizing enzyme, baclofen,mexiletine, sulfisoxazole and torasemide, or salt(s), prodrug(s) orsustained release formulation(s) thereof.
 19. The composition of claim18, said composition further comprising a compound selected frombaclofen, mexiletine, sulfisoxazole and torasemide or salt(s),prodrug(s) or sustained release formulation(s) thereof.
 20. Thecomposition of claim 19, said composition comprising: tadalafil,cinacalcet and mexiletine, or tadalafil, cinacalcet and baclofen, orsalt(s), prodrug(s), or sustained release formulation(s) thereof. 21.The composition of claim 20, said composition further comprising adopamine precursor selected from levodopa or melevodopa or salts,prodrugs or sustained release formulation thereof.
 22. The compositionof claim 20, said composition further comprising an inhibitor of adopamine metabolizing enzyme selected from selegiline or rasagiline, orany pharmaceutically acceptable salt(s), prodrug(s), or sustainedrelease formulation thereof.
 23. The composition of claim 20, saidcomposition further comprising an inhibitor of a dopamine metabolizingenzyme selected from carbidopa, benserazide, entacapone, tolcapone,selegiline and rasagiline, or any pharmaceutically acceptable salt(s),prodrug(s), or sustained release formulation thereof.
 24. Thecomposition of claim 18, said composition further comprising a dopaminereceptor agonist selected from the group consisting of talipexole,piribedil, rotigotine, bromocriptine, pergolide, cabergoline, lisuride,pramipexole, ropinirole and apomorphine or salts, prodrugs or sustainedrelease formulations thereof.